Method for the synthesis of phosphorus atom modified nucleic acids

ABSTRACT

Described herein are methods of syntheses of phosphorous atom-modified nucleic acids comprising chiral X-phosphonate moieties. The methods described herein provide backbone-modified nucleic acids in high diasteteomeric purity via an asymmetric reaction of an achiral molecule comprising a chemically stable H-phosphonate moiety with a nucleoside/nucleotide.

FIELD OF THE INVENTION

Described herein are methods of syntheses of phosphorous atom-modifiednucleic acids comprising chiral X-phosphonate moieties. The methodsdescribed herein provide backbone-modified nucleic acids in highdiastereomeric purity via an asymmetric reaction of an achiral moleculecomprising a chemically stable H-phosphonate moiety with anucleoside/nucleotide.

BACKGROUND OF THE INVENTION

Oligonucleotides are useful in therapeutic, diagnostic, research, andnew and nanomaterials applications. The use of natural sequences of DNAor RNA is limited by their stability to nucleases. Additionally, invitro studies have shown that the properties of antisense nucleotidessuch as binding affinity, sequence specific binding to the complementaryRNA, stability to nucleases are affected by the configurations of thephosphorous atoms. Therefore, there is a need in the field for methodsto produce oligonucleotides which are stereocontrolled at phosphorus andexhibit desired stability to degradation while retaining affinity forexogenous or endogenous complementary DNA/RNA sequences. There is a needfor these compounds to be easily synthesized on solid support or insolution, and to permit a wide range of synthetic modifications on thesugars or nucleobases of the oligonucleotide.

Described herein are stereocontrolled syntheses of phosphorousatom-modified polymeric and oligomeric nucleic acids, which in someembodiments, is performed on solid support.

SUMMARY OF THE INVENTION

In one aspect of the invention, a method for a synthesis of a nucleicacid is provided comprising a chiral X-phosphonate moiety comprisingreacting a molecule comprising an achiral H-phosphonate moiety and anucleoside comprising a 5′-OH moiety to form a condensed intermediate;and converting the condensed intermediate to the nucleic acid comprisinga chiral X-phosphonate moiety.

In some embodiments, the method wherein the step of reacting themolecule comprising an achiral H-phosphonate moiety and the nucleosidecomprising a 5′-OH moiety to form a condensed intermediate is a one-potreaction.

In some embodiments, the method provides a nucleic acid comprising achiral X-phosphonate moiety of Formula 1.

In some embodiments of the compound of Formula 1, R¹ is —OH, —SH,—NR^(d)R^(d), —N₃, halogen, hydrogen, alkyl, alkenyl, alkynyl,alkyl-Y¹—, alkenyl-Y¹—, alkynyl-Y¹—, heteroaryl-Y¹—, —P(O)(R^(e))₂,—HP(O)(R^(e)), —OR^(a) or —SR^(c). Y¹ is O, NR^(d), S, or Se. R^(a) is ablocking moiety. R^(c) is a blocking group. Each instance of R^(d) isindependently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substitutedsilyl, carbamate, —P(O)(R^(e))₂, or —HP(O)(R^(e)). Each instance ofR^(e) is independently hydrogen, alkyl, aryl, alkenyl, alkynyl,alkyl-Y²—, alkenyl-Y²—, alkynyl-Y²—, aryl-Y²—, or heteroaryl-Y²—, or acation which is Na⁺¹, Li⁺¹, or K⁺¹. Y² is O, NR^(d), or S. Each instanceof R² is independently hydrogen, —OH, —SH, —NR^(d)R^(d), —N₃, halogen,alkyl, alkenyl, alkynyl, alkyl-Y¹—, alkenyl-Y¹—, alkynyl-Y¹—, aryl-Y¹—,heteroaryl-Y¹—, —OR^(b), or —SR^(c), wherein R^(b) is a blocking moiety.Each instance of Ba is independently a blocked or unblocked adenine,cytosine, guanine, thymine, uracil or modified nucleobase. Each instanceof X is independently alkyl, alkoxy, aryl, alkylthio, acyl,—NR^(f)R^(f), alkenyloxy, alkynyloxy, alkenylthio, alkynylthio, —S⁻Z⁺,—Se⁻Z⁺, or —BH₃ ⁻Z⁺. Each instance of R^(f) is independently hydrogen,alkyl, alkenyl, alkynyl, or aryl. Z⁺ is ammonium ion, alkylammonium ion,heteroaromatic iminium ion, or heterocyclic iminium ion, any of which isprimary, secondary, tertiary or quaternary, or Z is a monovalent metalion. R³ is hydrogen, a blocking group, a linking moiety connected to asolid support or a linking moiety connected to a nucleic acid; and n isan integer of 1 to about 200.

In some embodiments of the method, each X-phosphonate moiety of thecompound of Formula 1 is more than 98% diastereomerically pure asdetermined by ³¹P NMR spectroscopy or reverse-phase HPLC. In someembodiments of the method, each X-phosphonate moiety has a R_(P)configuration. In other embodiments of the method, each X-phosphonatemoiety has a S_(P) configuration. In other embodiments of the method,each X-phosphonate independently has a R_(P) configuration or a S_(P)configuration.

In further embodiments of the method, the molecule comprising an achiralH-phosphonate moiety is a compound of Formula 2.

In Formula 2, R¹ is —NR^(d)R^(d), —N₃, halogen, hydrogen, alkyl,alkenyl, alkynyl, alkyl-Y¹—, alkenyl-Y¹—, alkynyl-Y¹—, aryl-Y¹—,heteroaryl-Y¹—, —P(O)(R^(e))₂, —HP(O)(R^(e)), —OR^(a), or —SR^(c). Y¹ isO, NR^(d), S, or Se. R^(a) is a blocking moiety. R^(c) is a blockinggroup. Each instance of R^(d) is independently hydrogen, alkyl, alkenyl,alkynyl, aryl, acyl, substituted silyl, carbamate, —P(O)(R^(e))₂, or—HP(O)(R^(e)). Each instance of R^(e) is independently alkyl, aryl,alkenyl, alkynyl, alkyl-Y²—, alkenyl-Y²—, alkynyl-Y²—, aryl-Y²—, orheteroaryl-Y²—. Y² is O, NR^(d), or S. R² is hydrogen, —NR^(d)R^(d), N₃,halogen, alkyl, alkenyl, alkynyl, alkyl-Y¹—, alkenyl-Y¹—, alkynyl-Y¹—,aryl-Y¹—, heteroaryl-Y¹—, —OR^(b), or —SR^(c), wherein R^(b) is ablocking moiety. Ba is a blocked or unblocked adenine, cytosine,guanine, thymine, uracil or modified nucleobase. Z⁺ is ammonium ion,alkylammonium ion, heteroaromatic iminium ion, or heterocyclic iminiumion, any of which is primary, secondary, tertiary or quaternary, or amonovalent metal ion.

In some embodiments of the method, the method further comprises a chiralreagent. In yet other embodiments of the method, the chiral reagent is acompound of Formula 3.

W₁ and W₂ are independently —NG⁵-, —O—, or —S—. G¹, G², G³, G⁴, and G⁵are independently hydrogen, alkyl, aralkyl, cycloalkyl, cycloalkylalkyl,heterocyclyl, hetaryl, or aryl, or two of G¹, G², G³, G⁴, and G⁵ are G⁶taken together form a saturated, partially unsaturated or unsaturatedcarbocyclic or heteroatom-containing ring of up to about 20 ring atomswhich is monocyclic or polycyclic, fused or unfused, and wherein no morethan four of G¹, G², G³, G⁴, and G⁵ are G⁶.

In some embodiments of the method, the nucleoside comprising a 5′-OHmoiety is a compound of Formula 4.

Each instance of R² is independently hydrogen, —NR^(d)R^(d), N₃,halogen, alkyl, alkenyl, alkynyl, alkyl-Y¹—, alkenyl-Y¹—, alkynyl-Y¹—,aryl-Y¹—, heteroaryl-Y¹—, —OR^(b), or —SR^(c), wherein R^(b) is ablocking moiety. Y¹ is O, NR^(d), S, or Se. R^(c) is a blocking group.Each instance of R^(d) is independently hydrogen, alkyl, alkenyl,alkynyl, aryl, acyl, substituted silyl, carbamate, —P(O)(R^(e))₂, or—HP(O)(R^(e)). Each instance of R^(e) is independently alkyl, aryl,alkenyl, alkynyl, alkyl-Y²—, alkenyl-Y²—, alkynyl-Y²—, aryl-Y², orheteroaryl-Y²—. Y² is O, NR^(d), or S. Each instance of Ba isindependently a blocked or unblocked adenine, cytosine, guanine,thymine, uracil or modified nucleobase. m is an integer of 0 to n−1. nis an integer of 1 to about 200. O_(A) is connected to a trityl moiety,a silyl moiety, an acetyl moiety, an acyl moiety, an aryl acyl moiety, alinking moiety connected to a solid support or a linking moietyconnected to a nucleic acid. J is O and D is H, or J is S, Se, or BH₃and D is a chiral ligand C_(i) or a moiety of Formula A.

In Formula A, W₁ and W₂ are independently NHG⁵, OH, or SH. A ishydrogen, acyl, aryl, alkyl, aralkyl, or a silyl moiety. G¹, G², G³, G⁴,and G⁵ are independently hydrogen, alkyl, aralkyl, cycloalkyl,cycloalkylalkyl, heterocyclyl, heteroaryl, or aryl, or two of G¹, G²,G³, G⁴, and G⁵ are G⁶ which taken together form a saturated, partiallyunsaturated or unsaturated carbocyclic or heteroatom-containing ring ofup to about 20 ring atoms which is monocyclic or polycyclic, fused orunfused and wherein no more than four of G¹, G², G³, G⁴, and G⁵ are G⁶.

In yet other embodiments of the method, the method further comprisesproviding a condensing reagent C_(R) whereby the molecule comprising anachiral H-phosphonate moiety is activated to react with the chiralreagent to form a chiral intermediate.

In further embodiments of the method, the condensing reagent C_(R) isAr₃PL₂, (ArO)₃PL₂,

Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, Z⁷, Z⁸, Z⁹ and Z¹⁰ are independently alkyl,aminoalkyl, cycloalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl,aryl, heteroaryl, alkyloxy, aryloxy, or heteroaryloxy, or wherein any ofZ² and Z³, Z⁵ and Z⁶, Z⁷ and Z⁸, Z⁸ and Z⁹, Z⁹ and Z⁷, or Z⁷ and Z⁸ andZ⁹ are taken together to form a 3 to 20 membered alicyclic orheterocyclic ring. Q⁻ is a counter anion, L is a leaving group, and w isan integer of 0 to 3. Ar is aryl, heteroaryl, and/or one of Ar group isattached to the polymer support. In some embodiments of the method, thecounter ion of the condensing reagent C_(R) is Cl⁻, Br⁻, BF₄ ⁻, PF₆ ⁻,TfO⁻, Tf₂N⁻, AsF₆ ⁻, ClO₄ ⁻, or SbF₆ ⁻, wherein Tf is CF₃SO₂. In otherembodiments of the method, the leaving group of the condensing reagentC_(R) is F, Cl, Br, I, 3-nitro-1,2,4-triazole, imidazole, alkyltriazole,tetrazole, pentafluorobenzene, or 1-hydroxybenzotriazole.

In some embodiments of the method, the condensing reagent is phosgene,trichloromethyl chloroformate, bis(trichloromethyl)carbonate (BTC),oxalyl chloride, Ph₃PCl₂, (PhO)₃PCl₂,N,N′-bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BopCl),1,3-dimethyl-2-(3-nitro-1,2,4-triazol-1-yl)-2-pyrrolidin-1-yl-1,3,2-diazaphospholidiniumhexafluorophosphate (MNTP), or3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphoniumhexafluorophosphate (PyNTP).

In a further embodiment of the method, the method further comprisesproviding an activating reagent A_(R). In one embodiment, the activatingreagent A_(R) is

wherein Z¹¹, Z¹², Z¹³, Z¹⁴, Z¹⁵, Z¹⁶, Z¹⁷, Z¹⁸, Z¹⁹, Z²⁰, and Z²¹ areindependently hydrogen, alkyl, aminoalkyl, cycloalkyl, heterocyclic,cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy,or heteroaryloxy, or wherein any of Z¹¹ and Z¹², Z¹¹ and Z¹³, Z¹¹ andZ¹⁴, Z¹² and Z¹³, Z¹² and Z¹⁴, Z¹³ and Z¹⁴, Z¹⁵ and Z¹⁶, Z¹⁵ and Z¹⁷,Z¹⁶ and Z¹⁷, Z¹⁸ and Z¹⁹, or Z²⁰ and Z²¹ are taken together to form a 3to 20 membered alicyclic or heterocyclic ring, or to form 5 or 20membered aromatic ring; and Q⁻ is a counter ion. In an embodiment, thecounter ion of the activating reagent A_(R) is Cl⁻, Br⁻, BF₄ ⁻, PF₆ ⁻,TfO⁻, Tf₂N⁻, AsF₆ ⁻, ClO₄ ⁻, or SbF₆ ⁻, wherein Tf is CF₃SO₂. In oneembodiment, the activating reagent A_(R) is imidazole,4,5-dicyanoimidazole (DCI), 4,5-dichloroimidazole, 1-phenylimidazoliumtriflate (PhIMT), benzimidazolium triflate (BIT), benztriazole,3-nitro-1,2,4-triazole (NT), tetrazole, 5-ethylthiotetrazole,5-(4-nitrophenyl)tetrazole, N-cyanomethylpyrrolidinium triflate (CMPT),N-cyanomethylpiperidinium triflate, N-cyanomethyldimethylammoniumtriflate. In another embodiment, the activating reagent A_(R) is4,5-dicyanoimidazole (DCI), 1-phenylimidazolium triflate (PhIMT),benzimidazolium triflate (BIT), 3-nitro-1,2,4-triazole (NT), tetrazole,or N-cyanomethylpyrrolidinium triflate (CMPT).

In an embodiment, the activating reagent A_(R) isN-cyanomethylpyrrolidinium triflate (CMPT).

In some embodiments of the method, the reaction is performed in anaprotic organic solvent. In other embodiments of the method, the solventis acetonitrile, pyridine, tetrahydrofuran, or dichloromethane. In otherembodiments of the method, when the aprotic organic solvent is notbasic, a base is present in the reacting step. In some embodiments ofthe method, the base is pyridine, quinoline, or N,N-dimethylaniline. Insome embodiments of the method, the base is

wherein Z²² and Z²³ are independently alkyl, aminoalkyl, cycloalkyl,heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl,alkyloxy, aryloxy, or heteroaryloxy, or wherein any of Z²² and Z²³ aretaken together to form a 3 to 10 membered alicyclic or heterocyclicring. In some embodiments of the method, the base isN-cyanomethylpyrrolidine. In some embodiments of the method, the aproticorganic solvent is anhydrous. In other embodiments of the method, theanhydrous aprotic organic solvent is freshly distilled. In yet otherembodiments of the method, the freshly distilled anhydrous aproticorganic solvent is pyridine. In another embodiment of the method, thefreshly distilled anhydrous aprotic organic solvent is acetonitrile.

In some embodiments of the method, the step of converting the condensedintermediate to a compound of Formula 1 comprises: modifying thecondensed intermediate to produce a compound of Formula 5.

In Formula 5, R¹ is —NR^(d)R^(d), —N₃, halogen, hydrogen, alkyl,alkenyl, alkynyl, alkenyl-Y¹—, alkynyl-Y¹—, heteroaryl-Y¹—,—P(O)(R^(e))₂, —HP(O)(R^(e)), —OR^(a), or —SR^(c). Y¹ is O, NR^(d), S,or Se. R^(a) is a blocking moiety. R^(c) is a blocking group. Eachinstance of R^(d) is independently hydrogen, alkyl, alkenyl, alkynyl,aryl, acyl, substituted silyl, carbamate, —P(O)(R^(e))₂, or—HP(O)(R^(e)). Each instance of R^(e) is independently alkyl, aryl,alkenyl, alkynyl, alkyl-Y²—, alkenyl-Y²—, alkynyl-Y²—, aryl-Y²—, orheteroaryl-Y²—. Y² is O, NR^(d), or S. Each instance of R² isindependently hydrogen, —NR^(d)R^(d), N₃, halogen, alkyl, alkenyl,alkynyl, alkyl-Y¹—, alkenyl-Y¹—, alkynyl-Y¹—, aryl-Y¹—, heteroaryl-Y¹—,—OR^(b), or —SR^(c), wherein R^(b) is a blocking moiety. Each instanceof Ba is independently a blocked or unblocked adenine, cytosine,guanine, thymine, uracil, or modified nucleobase. Each instance of J isS, Se, or BH₃. v is an integer of 1. O_(A) is connected to a linkingmoiety connected to a solid support or a linking moiety connected to anucleic acid. A is an acyl, aryl, alkyl, aralkyl, or silyl moiety. G¹,G², G³, G⁴, and G⁵ are independently hydrogen, alkyl, aralkyl,cycloalkyl, cycloalkylalkyl, heterocyclyl, heteroaryl, or aryl, or twoof G¹, G², G³, G⁴, and G⁵ are G⁶ which taken together form a saturated,partially unsaturated or unsaturated carbocyclic orheteroatom-containing ring of up to about 20 ring atoms which ismonocyclic or polycyclic, fused or unfused and wherein no more than fourof G¹, G², G³, G⁴, and G⁵ are G⁶.

In some embodiments of the method, the step of converting the condensedintermediate to a compound of Formula 1 comprises: capping the condensedintermediate and modifying the capped condensed intermediate to producea compound of Formula 5.

In Formula 5, R¹ is —NR^(d)R^(d), —N₃, halogen, hydrogen, alkyl,alkenyl, alkynyl, alkyl-Y¹—, alkenyl-Y¹—, alkynyl-Y¹—, aryl-Y¹—,heteroaryl-Y¹—, —P(O)(R^(e))₂, —HP(O)(R^(e)), —OR^(a), or —SR^(c). Y¹ isO, NR^(d), S, or Se. R^(a) is a blocking moiety. R^(c) is a blockinggroup. Each instance of R^(d) is independently hydrogen, alkyl, alkenyl,alkynyl, aryl, acyl, substituted silyl, carbamate, —P(O)(R^(e))₂, or—HP(O)(R^(e)). Each instance of R^(e) is independently alkyl, aryl,alkenyl, alkynyl, alkyl-Y²—, alkenyl-Y²—, alkynyl-Y²—, aryl-Y²—, orheteroaryl-Y²—. Y² is O, NR^(d), or S. Each instance of R² isindependently hydrogen, —NR^(d)R^(d), N₃, halogen, alkyl, alkenyl,alkynyl, alkyl-Y¹—, alkenyl-Y¹—, alkynyl-Y¹—, aryl-Y¹—, heteroaryl-Y¹—,—OR^(b), or —SR^(c), wherein R^(b) is a blocking moiety. Each instanceof Ba is independently a blocked or unblocked adenine, cytosine,guanine, thymine, uracil, or modified nucleobase. Each instance of J isS, Se, or BH₃. v is an integer of 2 to n−1. O_(A) is connected to alinking moiety connected to a solid support or a linking moietyconnected to a nucleic acid. A is an acyl, aryl, alkyl, aralkyl, orsilyl moiety. G¹, G², G³, G⁴, and G⁵ are independently hydrogen, alkyl,aralkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heteroaryl, or aryl,or two of G¹, G², G³, G⁴, and G⁵ are G⁶ which taken together form asaturated, partially unsaturated or unsaturated carbocyclic orheteroatom-containing ring of up to about 20 ring atoms which ismonocyclic or polycyclic, fused or unfused and wherein no more than fourof G¹, G², G³, G⁴, and G⁵ are G⁶.

In some embodiments of the method, the method further comprises thesteps of: (a) deblocking R¹ of the compound of Formula 5 to produce acompound of Formula 4 wherein m is at least 1, J is S, Se, or BH₃ and Dis a moiety of Formula A; (b) reacting the compound of Formula 4 usingthe method of claim 10 wherein the step of converting the condensedintermediate comprises capping the condensed intermediate and modifyingthe capped condensed intermediate to produce a compound of Formula 5wherein v is greater than 2 and less than about 200; and (c) optionallyrepeating steps (a) and (b) to form a compound of Formula 5 wherein v isgreater than 3 and less than about 200.

In other embodiments of the method, the method further comprises thestep of converting the compound of Formula 5 to the compound of Formula1 wherein each Ba moiety is unblocked. R¹ is —OH, —SH, —NR^(d)R^(d),—N₃, halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Y¹—, alkenyl-Y¹—,alkynyl-Y¹—, aryl-Y¹—, heteroaryl-Y¹—, —P(O)(R^(e))₂, —HP(O)(R^(e)),—OR^(a) or —SR^(c). Y¹ is O, NR^(d), S, or Se. R^(a) is a blockingmoiety. R^(c) is a blocking group. Each instance of R^(d) isindependently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substitutedsilyl, carbamate, —P(O)(R^(e))₂, or —HP(O)(R^(e)). Each instance ofR^(e) is independently hydrogen, alkyl, aryl, alkenyl, alkynyl,alkyl-Y²—, alkenyl-Y²—, alkynyl-Y²—, aryl-Y²—, or heteroaryl-Y²—, or acation which is Na⁺¹, Li⁺¹, or K⁺¹. Y² is O, NR^(d), or S. Each instanceof R² is independently hydrogen, —OH, —SH, —NR^(d) _(R) ^(d), —N₃,halogen, alkyl, alkenyl, alkynyl, alkyl-Y¹—, alkenyl-Y¹—, alkynyl-Y¹—,aryl-Y¹—, heteroaryl-Y¹—, —OR^(b), or —SR^(c), wherein R^(b) is ablocking moiety. R³ is H. Each instance of X is independently —S⁻Z⁺,—Se⁻Z⁺, or —BH₃ ⁻Z⁺. Z⁺ is ammonium ion, alkylammonium ion,heteroaromatic iminium ion, or heterocyclic iminium ion, any of which isprimary, secondary, tertiary or quaternary, or Z is a monovalent metalion.

In some embodiments of the method, the step of converting the condensedintermediate to a compound of Formula 1 comprises acidifying thecondensed intermediate to produce a compound of Formula 4, wherein m isat least one, J is O, and D is H. In some embodiments of the method, thecondensed intermediate comprises a moiety of Formula A′.

A is hydrogen and G¹ and G² are independently alkyl, aralkyl,cycloalkyl, cycloalkylalkyl, heteroaryl, or aryl and G³, G⁴, and G⁵ areindependently hydrogen, alkyl, aralkyl, cycloalkyl, cycloalkylalkyl,heterocyclyl, heteroaryl, or aryl, or two of G¹, G², G³, G⁴, and G⁵ areG⁶ which taken together form a saturated, partially unsaturated orunsaturated carbocyclic or heteroatom-containing ring of up to about 20ring atoms which is monocyclic or polycyclic, fused or unfused andwherein no more than four of G¹, G², G³, G⁴, and G⁵ are G⁶.

In some embodiments of the method, the method further comprises: (a)reacting the compound of Formula 4 wherein m is at least one, J is O,and D is H, using the method of claim 10 wherein the step of convertingthe condensed intermediate to a compound of Formula 1 comprisesacidifying the condensed intermediate to produce a compound of Formula 4wherein m is at least 2 and less than about 200; J is O, and D is H, and(b) optionally repeating step (a) to produce a compound of Formula 4wherein m is greater than 2 and less than about 200.

In some embodiments of the method, the acidifying comprises adding anamount of a Brønsted or Lewis acid effective to convert the condensedintermediate into the compound of Formula 4 without removing purine orpyrimidine moieties from the condensed intermediate. In otherembodiments of the method, the acidifying comprises adding 1%trifluoroacetic acid in an organic solvent, 3% dichloroacetic acid in anorganic solvent, or 3% trichloroacetic acid in an organic solvent. Inyet other embodiments of the method, the acidifying further comprisesadding a cation scavenger. In some embodiments of the method, the cationscavenger is triethylsilane or triisopropylsilane.

In some embodiments of the method, the step of converting the condensedintermediate to a compound of Formula 1 further comprises deblocking R¹prior to the step of acidifying the condensed intermediate.

In other embodiments of the method, the method further comprises thestep of modifying the compound of Formula 4 to introduce an X moietythereby producing a compound of Formula 1 wherein R³ is a blocking groupor a linking moiety connected to a solid support.

In yet other embodiments of the method, the method further comprisestreating an X-modified compound to produce a compound of Formula 1wherein R¹ is —OH, —SH, —NR^(d)R^(d), —N₃, halogen, hydrogen, alkyl,alkenyl, alkynyl, alkyl-Y¹—, alkenyl-Y¹—, aryl-Y¹—, heteroaryl-Y¹—,—P(O)(R^(e))₂, —HP(O)(R^(e)), —OR^(a) or —SR^(c). Y¹ is O, NR^(d), S, orSe. R^(a) is a blocking moiety. R^(c) is a blocking group. Each instanceof R^(d) is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl,substituted silyl, carbamate, —P(O)(R^(c))₂, or —HP(O)(R^(e)). Eachinstance of R^(e) is independently hydrogen, alkyl, aryl, alkenyl,alkynyl, alkyl-Y²—, alkenyl-Y²—, alkynyl-Y²—, aryl-Y²—, orheteroaryl-Y²—, or a cation which is Na⁺¹, Li⁺¹, or K⁺¹. Y² is O,NR^(d), or S. Each instance of R² is independently hydrogen, —OH, —SH,—NR^(d)R^(d), —N₃, halogen, alkyl, alkenyl, alkynyl, alkyl-Y¹—,alkenyl-Y¹—, alkynyl-Y¹—, aryl-Y¹—, heteroaryl-Y¹—, —OR^(b), or —SR^(c),wherein R^(b) is a blocking moiety. Each Ba moiety is unblocked. R³ isH. Each instance of X is independently alkyl, alkoxy, aryl, alkylthio,acyl, —NR^(f)R^(f), alkenyloxy, alkynyloxy, alkenylthio, alkynylthio,—S⁻Z⁺, —Se⁻Z⁺, or —BH₃ ⁻Z⁺. Each instance of R^(f) is independentlyhydrogen, alkyl, alkenyl, alkynyl, or aryl. Z⁺ is ammonium ion,alkylammonium ion, heteroaromatic iminium ion, or heterocyclic iminiumion, any of which is primary, secondary, tertiary or quaternary, or Z isa monovalent metal ion. n is greater than 1 and less than about 200.

In some embodiments of the method, the modifying step is performed usinga boronating agent, a sulfur electrophile, or a selenium electrophile.

In some embodiments of the method, the sulfur electrophile is a compoundhaving one of the following formulas:

S₈ (Formula B), Z²⁴—S—S—Z²⁵, or Z²⁴—S—X—Z²⁵.

Z²⁴ and Z²⁵ are independently alkyl, aminoalkyl, cycloalkyl,heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl,alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide, or thiocarbonyl,or Z²⁴ and Z²⁵ are taken together to form a 3 to 8 membered alicyclic orheterocyclic ring, which may be substituted or unsubstituted; X is SO₂,O, or NR^(f); and R^(f) is hydrogen, alkyl, alkenyl, alkynyl, or aryl.

In some embodiments of the method, the sulfur electrophile is a compoundof Formula B, C, D, E, or F:

In some embodiments of the method, the selenium electrophile is acompound having one of the following formulas:

Se (Formula G), Z²⁶—Se—Se—Z²⁷, or Z²⁶—Se—X—Z²⁷

Z²⁶ and Z²⁷ are independently alkyl, aminoalkyl, cycloalkyl,heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl,alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide, or thiocarbonyl,or Z²⁶ and Z²⁷ are taken together to form a 3 to 8 membered alicyclic orheterocyclic ring, which may be substituted or unsubstituted; X is SO₂,S, O, or NR^(f); and R^(f) is hydrogen, alkyl, alkenyl, alkenyl, oraryl.

In some embodiments of the method, the selenium electrophile is acompound of Formula G, H, I, J, K, or L.

In some embodiments of the method, the boronating agent isborane-N,N-diisopropylethylamine (BH₃.DIPEA), borane-pyridine (BH₃.Py),borane-2-chloropyridine (BH₃.CPy), borane-aniline (BH₃.An),borane-tetrahydrofurane (BH₃.THF), or borane-dimethylsulfide (BH₃.Me₂S).

In some embodiments of the method, the modifying step is performed usinga silylating reagent followed by a sulfur electrophile, a seleniumelectrophile, a boronating agent, an alkylating agent, an aldehyde, oran acylating agent.

In some embodiments of the method, the silylating reagent ischlorotrimethylsilane (TMS-Cl), triisopropylsilylchloride (TIPS-Cl),t-butyldimethylsilylchloride (TBDMS-Cl), t-butyldiphenylsilylchloride(TBDPS-Cl), 1,1,1,3,3,3-hexamethyldisilazane (HMDS),N-trimethylsilyldimethylamine (TMSDMA), N-trimethylsilyldiethylamine(TMSDEA), N-trimethylsilylacetamide (TMSA),N,O-bis(trimethylsilyl)acetamide (BSA), orN,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA).

In some embodiments of the method, the sulfur electrophile is a compoundhaving one of the following formulas: S₈ (Formula B), Z²⁴—S—S—Z²⁵, orZ²⁴—S—X—Z²⁵, wherein Z²⁴ and Z²⁵ are independently alkyl, aminoalkyl,cycloalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl,heteroaryl, alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide, orthiocarbonyl, or Z²⁴ and Z²⁵ are taken together to form a 3 to 8membered alicyclic or heterocyclic ring, which may be substituted orunsubstituted; X is SO₂, O, or NR^(f); and R^(f) is hydrogen, alkyl,alkenyl, alkynyl, or aryl.

In some embodiments of the method, the sulfur electrophile is a compoundof Formula B, C, D, E, or F:

In some embodiments of the method, the selenium electrophile is acompound having one of the following formulas: Se (Formula G),Z²⁶—Se—Se—Z²⁷, or Z²⁶—Se—X—Z²⁷, wherein Z²⁶ and Z²⁷ are independentlyalkyl, aminoalkyl, cycloalkyl, heterocyclic, cycloalkylalkyl,heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy, heteroaryloxy,acyl, amide, imide, or thiocarbonyl, or Z²⁶ and Z²⁷ are taken togetherto form a 3 to 8 membered alicyclic or heterocyclic ring, which may besubstituted or unsubstituted; X is SO₂, S, O, or NR^(f); and R^(f) ishydrogen, alkyl, alkenyl, alkynyl, or aryl.

In some embodiments of the method, the selenium electrophile is acompound of Formula G, H, I, J, K, or L:

In some embodiments of the method, the boronating agent isborane-N,N-diisopropylethylamine (BH₃.DIPEA), borane-pyridine (BH₃.Py),borane-2-chloropyridine (BH₃.CPy), borane-aniline (BH₃.An),borane-tetrahydrofurane (BH₃.THF), or borane-dimethylsulfide (BH₃.Me₂S).

In some embodiments of the method, the alkylating agent is an alkylhalide, alkenyl halide, alkynyl halide, alkyl sulfonate, alkenylsulfonate, or alkynyl sulfonate. In other embodiments of the method, thealdehyde is (para)-formaldehyde, alkyl aldehyde, alkenyl aldehyde,alkynyl aldehyde, or aryl aldehyde.

In some embodiments of the method, the acylating agent is a compound ofFormula M or N.

G⁷ is alkyl, cycloalkyl, heterocyclic, cycloalkylalkyl,heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy, or heteroaryloxy;and M is F, Cl, Br, I, 3-nitro-1,2,4-triazole, imidazole, alkyltriazole,tetrazole, pentafluorobenzene, or 1-hydroxybenzotriazole.

In some embodiments of the method, the modifying step is performed byreacting with a halogenating reagent followed by reacting with anucleophile. In some embodiments of the method, the halogenating reagentis CCl₄, CBr₄, Cl₂, Br₂, I₂, sulfuryl chloride (SO₂Cl₂), phosgene,bis(trichloromethyl)carbonate (BTC), sulfur monochloride, sulfurdichloride, chloramine, CuCl₂, N-chlorosuccinimide (NCS), CI₄,N-bromosuccinimide (NBS), or N-iodosuccinimide (NIS). In otherembodiments of the method, the nucleophile is NR^(f)R^(f)H, R^(f)OH, orR^(f)SH, wherein R^(f) is hydrogen, alkyl, alkenyl, alkenyl, or aryl,and at least one of R^(f) of NR^(f)R^(f)H is not hydrogen.

In some embodiments of the method, the chiral reagent is the compound ofFormula 3 wherein W₁ is NHG⁵ and W₂ is OH. In some embodiments of themethod, the chiral reagent is Formula O or Formula P.

In some embodiments of the method, the chiral reagent is Formula Q orFormula R.

In some embodiments of the method, R^(a) is substituted or unsubstitutedtrityl or substituted silyl. In other embodiments of the method, whereinR^(a) is substituted or unsubstituted trityl or substituted silyl. Inother embodiments of the method, R^(b) is substituted or unsubstitutedtrityl, substituted silyl, acetyl, acyl, or substituted methyl ether.

In some embodiments of the method, R³ is a blocking group which issubstituted trityl, acyl, substituted silyl, or substituted benzyl. Inother embodiments of the method, R³ is a linking moiety connected to asolid support.

In some embodiments of the method, the blocking group of the Ba moietyis a benzyl, acyl, formyl, dialkylformamidinyl, isobutyryl,phenoxyacetyl, or trityl moiety, any of which may be unsubstituted orsubstituted. In some embodiments of the method, R¹ is —N₃, —NR^(d)R^(d),alkynyloxy, or —OH. In some embodiments of the method, R¹ is —N₃,—NR^(d)R^(d), alkynyloxy, or —OH. In other embodiments of the method, R²is —NR^(d)R^(d), alkyl, alkenyl, alkynyl, alkyl-Y¹—, alkenyl-Y¹—,alkynyl-Y¹—, aryl-Y¹—, or heteroaryl-Y¹—, and is substituted withfluorescent or biomolecule binding moieties. In yet other embodiments ofthe method, R² is —NR^(d)R^(d), alkyl, alkenyl, alkynyl, alkyl-Y¹—,alkenyl-Y¹—, alkynyl-Y¹—, aryl-Y¹—, or heteroaryl-Y¹—, and issubstituted with fluorescent or biomolecule binding moieties.

In some embodiments of the method, the substituent on R² is afluorescent moiety. In other embodiments of the method, the substituenton R² is biotin or avidin. In yet other embodiments of the method, thesubstituent on R² is a fluorescent moiety. In some embodiments of themethod, the substituent on R² is biotin or avidin. In other embodimentsof the method, R² is —OH, —N₃, hydrogen, halogen, alkoxy, or alkynyloxy.In yet other embodiments of the method, R² is —OH, —N₃, hydrogen,halogen, alkoxy, or alkynyloxy.

In some embodiments of the method, Ba is 5-bromouracil, 5-iodouracil, or2,6-diaminopurine. In other embodiments of the method, Ba is modified bysubstitution with a fluorescent or biomolecule binding moiety. In yetother embodiments of the method, Ba is modified by substitution with afluorescent or biomolecule binding moiety. In some embodiments of themethod, the substituent on Ba is a fluorescent moiety. In otherembodiments of the method, the substituent on Ba is biotin or avidin. Inyet other embodiments of the method, the substituent on Ba is afluorescent moiety. In some embodiments of the method, the substituenton Ba is biotin or avidin.

In some embodiments of the method, Z is pyridinium ion, triethylammoniumion, N,N-diisopropylethylammonium ion,1,8-diazabicyclo[5.4.0]undec-7-enium ion, sodium ion, or potassium ion.In other embodiments of the method, Z is pyridinium ion,triethylammonium ion, N,N-diisopropylethylammonium ion,1,8-diazabicyclo[5.4.0]undec-7-enium ion, sodium ion, or potassium ion.In some embodiments of the method, X is alkyl, alkoxy, —NR^(f)R^(f),—S⁻Z⁺, or —BH₃ ⁻Z⁺. In other embodiments of the method, X is alkyl,alkoxy, —NR^(f)R^(f), —S⁻Z⁺, or —BH₃ ⁻Z⁺.

In an embodiment of the method, the sulfur electrophile is Formula F,Formula E or Formula B. In some embodiments of the method, the sulfurelectrophile is Formula F, Formula E or Formula B. In other embodimentsof the method, the selenium electrophile is Formula G or Formula L. Inyet other embodiments of the method, the selenium electrophile isFormula G or Formula L. In some embodiments of the method, theboronating agent is borane-N,N-diisopropylethylamine (BH₃.DIPEA),borane-2-chloropyridine (BH₃.CPy), borane-tetrahydrofurane (BH₃.THF), orborane-dimethylsulfide (BH₃.Me₂S). In other embodiments of the method,the halogenating agent is CCl₄, CBr₄, Cl₂, sulfuryl chloride (SO₂Cl₂),or N-chlorosuccinimide (NCS). In yet other embodiments of the method,the condensing reagent is bis(trichloromethyl)carbonate (BTC), Ph₃PCl₂,or N,N′-bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BopCl).

In another aspect of the invention, a method is provided of identifyingor detecting a target molecule in a sample, the method comprising:contacting a sample suspected of containing a target molecule with anucleic acid sensor molecule of Formula 1, synthesized according to themethods of the invention, wherein a change in a signal generated by asignal generating unit indicates the presence of said target in saidsample. The nucleic acid sensor molecule binds specifically with thetarget molecule. In some embodiments there is a plurality of nucleicacid sensor molecules. In some embodiments, the plurality of nucleicacid sensor molecules comprises nucleic acid sensor molecules which bindspecifically to differing target molecules. In some instances, themethod further comprises quantifying the change in signal generated bythe signal generating unit to quantify the amount of target molecule inthe sample. The signal generating unit detects any sort of signal,including but not limited to fluorescence, surface plasmon resonance,fluorescence quenching, chemiluminescence, interferometry, or refractiveindex detection.

The sample to be detected is an environmental sample, biohazardmaterial, organic sample, drug, toxin, flavor, fragrance, or biologicalsample. The biological sample is a cell, cell extract, cell lysate,tissue, tissue extract, bodily fluid, serum, blood or blood product. Insome embodiments of the method, the presence of the target moleculeindicates the presence of a pathological condition. In some embodimentsof the method, the presence of the target molecule indicates thepresence of a desirable molecule.

In another aspect of the invention, a method is provided of amplifyingdesired regions of nucleic acid from a nucleic acid template comprising:(a) providing a plurality of first PCR primers having a region of fixednucleotide sequence complementary to a consensus sequence of interest;(b) providing a plurality of second PCR primers, (c) amplifying thenucleic acid template via the PCR using the plurality of first PCRprimers and the plurality of second PCR primers under conditions whereina subset of the plurality of first primers binds to the consensussequence of interest substantially wherever it occurs in the template,and a subset of the plurality of second primers binds to the template atlocations removed from the first primers such that nucleic acid regionsflanked by the first primer and the second primer are specificallyamplified, and wherein the plurality of first PCR primers and/or theplurality of second PCT primers are nucleic acid molecules of Formula 1which are produced according to the methods of the invention.

In some embodiments, the template is genomic DNA. In some embodiments,the template is eukaryotic genomic DNA. In some embodiments, thetemplate is human genomic DNA. In some embodiments, the template isprokaryotic DNA. In some embodiments, the template is DNA which is acloned genomic DNA, a subgenomic region of DNA, a chromosome, or asubchromosomal region. In some embodiments, the template is RNA.

INCORPORATION BY REFERENCE

All publications and patent applications disclosed herein in thisspecification are herein incorporated by reference in their entirety tothe same extent as if each individual publication or patent applicationwas specifically and individually indicated to be incorporated byreference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1. ¹H NMR spectrum of (S_(P))-4tt (CDCl₃)

FIG. 2. ³¹P NMR spectrum of (S_(P))-4tt (CDCl₃)

FIG. 3. ¹H NMR spectrum of (R_(P))-4tt (CDCl₃)

FIG. 4. ³¹P NMR spectrum of (R_(P))-4tt (CDCl₃)

FIG. 5A. Crude UPLC® profile of (S_(P))-5tt

FIG. 5B. Crude UPLC® profile of (S_(P))-5tt using BTC in place ofPh3PCl2

FIG. 6A. Crude UPLC® profile of (S_(P))-5tt

FIG. 6B. Crude UPLC® profile of (S_(P))-5tt using BTC in place ofPH3PCL2

FIG. 7A. Crude UPLC® profile of (R_(P))-5tt

FIG. 7B. Crude UPLC® profile of (R_(P))-5tt

FIG. 8. Crude UPLC® profile of (R_(P))-5tt

FIG. 9A. Crude UPLC® profile of (S_(P))-5ct

FIG. 9B. Crude UPLC® profile of (S_(P))-5ct

FIG. 10A. Crude UPLC® profile of (R_(P))-5ct

FIG. 10B. Crude UPLC® profile of (R_(P))-5ct

FIG. 11. Crude UPLC® profile of (S_(P))-5at

FIG. 12. Crude UPLC® profile of (R_(P))-5at

FIG. 13. Crude UPLC® profile of (S_(P))-5gt

FIG. 14. Crude UPLC® profile of (R_(P))-5gt

FIG. 15A. Crude UPLC® profile of (S_(P))-5tt

FIG. 15B. Crude UPLC® profile of (S_(P))-5tt

FIG. 16A. Crude UPLC® profile of (S_(P))-5tt

FIG. 16B. Crude UPLC® profile of (S_(P))-5tt

FIG. 17A. Crude UPLC® profile of (R_(P))-5tt

FIG. 17B. Crude UPLC® profile of (R_(P))-5tt

FIG. 18. Crude UPLC® profile of (S_(P))-5ct

FIG. 19. Crude UPLC® profile of (R_(P))-5ct.

FIG. 20A. Crude UPLC® profile of (S_(P))-5at

FIG. 20B. Crude UPLC® profile of (S_(P))-5at

FIG. 21. Crude UPLC® profile of (S_(P))-5at

FIG. 22A. Crude UPLC® profile of (R_(P))-5at

FIG. 22B. Crude UPLC® profile of (R_(P))-5at

FIG. 23. Crude UPLC® profile of (S_(P))-5gt

FIG. 24. Crude UPLC® profile of (R_(P))-5gt

FIG. 25. Crude UPLC® profile of (S_(P))-7tt

FIG. 26. Crude UPLC® profile of (R_(P))-7tt

FIG. 27. Crude UPLC® profile of (S_(P))-8tt

FIG. 28. Crude UPLC® profile of (R_(P))-8tt

FIG. 29A. Crude UPLC® profile of All-(S_(P))-[T_(PS)]₃T

FIG. 29B. MALDI TOF-MS spectrum of All-(S_(P))-[T_(PS)]₃T

FIG. 30A. Crude UPLC® profile of (S_(P), R_(P), S_(P))-[T_(PS)]₃T

FIG. 30B. MALDI TOF-MS spectrum of (S_(P), R_(P), S_(P))-[T_(PS)]₃T

FIG. 31A. Crude UPLC® profile of (R_(P), S_(P), R_(P))-[T_(PS)]₃T

FIG. 31B. MALDI TOF-MS spectrum of (R_(P), S_(P), R_(P))-[T_(PS)]₃T

FIG. 32A. Crude UPLC® profile of All-(R_(P))-[T_(PS)]₃T

FIG. 32B. MALDI TOF-MS spectrum of All-(R_(P))-[T_(PS)]₃T

FIG. 33A. Crude UPLC® profile of (S_(P))-9u_(M)u

FIG. 33B. MALDI TOF-MS spectrum of (S_(P))-9u_(M)u

FIG. 34A. Crude UPLC® profile of (R_(P))-9uMu

FIG. 34B. MALDI TOF-MS spectrum of (R_(P))-9u_(M)u

FIG. 35A. Crude UPLC® profile of (S_(P))-10uFu

FIG. 35B. MALDI TOF-MS spectrum of (S_(P))-10u_(F)u

FIG. 36A. Crude UPLC® profile of (R_(P))-10uFu

FIG. 36B. MALDI TOF-MS spectrum of (R_(P))-10u_(F)u

FIG. 37A. Crude UPLC® profile of (S_(P))-11nt

FIG. 37B. MALDI TOF-MS spectrum of (S_(P))-11nt

FIG. 38A. Crude UPLC® profile of (R_(P))-11nt

FIG. 38B. MALDI TOF-MS spectrum of (R_(P))-11nt

DETAILED DESCRIPTION OF THE INVENTION Definitions.

Unless otherwise stated, the following terms used in this application,including the specification and claims, have the definitions givenbelow. It must be noted that, as used in the specification and theappended claims, the singular forms “a” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Unlessotherwise indicated, conventional methods of mass spectroscopy, NMR,HPLC, protein chemistry, biochemistry, recombinant DNA techniques andpharmacology are employed. In this application, the use of “or” or “and”means “and/or” unless stated otherwise. Furthermore, use of the term“including” as well as other forms, such as “include”, “includes” and“included” is not limiting.

The term “nucleic acid” encompasses poly- or oligo-ribonucleotides (RNA)and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA derived fromN-glycosides or C-glycosides of nucleobases and/or modified nucleobases;nucleic acids derived from sugars and/or modified sugars; and nucleicacids derived from phosphate bridges and/or modified phosphorous-atombridges. The term encompasses nucleic acids containing any combinationsof nucleobases, modified nucleobases, sugars, modified sugars, phosphatebridges or modified phosphorous atom bridges. Examples include, and arenot limited to, nucleic acids containing ribose moieties, the nucleicacids containing deoxy-ribose moieties, nucleic acids containing bothribose and deoxyribose moieties, nucleic acids containing ribose andmodified ribose moieties. The prefix poly-refers to a nucleic acidcontaining about 1 to about 10,000 nucleotide monomer units and whereinthe prefix oligo-refers to a nucleic acid containing about 1 to about200 nucleotide monomer units.

The term “nucleobase” refers to the parts of nucleic acids that areinvolved in the hydrogen-bonding that binds one nucleic acid strand toanother complementary strand in a sequence specific manner. The mostcommon naturally-occurring nucleobases are adenine (A), guanine (G),uracil (U), cytosine (C), and thymine (T).

The term “modified nucleobase” refers to a moiety that can replace anucleobase. The modified nucleobase mimics the spatial arrangement,electronic properties, or some other physicochemical property of thenucleobase and retains the property of hydrogen-bonding that binds onenucleic acid strand to another in a sequence specific manner. A modifiednucleobase can pair with all of the five naturally occurring bases(uracil, thymine, adenine, cytosine, or guanine) without substantiallyaffecting the melting behavior, recognition by intracellular enzymes oractivity of the oligonucleotide duplex.

The term “nucleoside” refers to a moiety wherein a nucleobase or amodified nucleobase is covalently bound to a sugar or modified sugar.

The term “sugar” refers to a monosaccharide in closed and/or open form.Sugars include, but are not limited to, ribose, deoxyribose,pentofuranose, pentopyranose, and hexopyranose moieties.

The term “modified sugar” refers to a moiety that can replace a sugar.The modified sugar mimics the spatial arrangement, electronicproperties, or some other physicochemical property of a sugar.

The term “nucleotide” refers to a moiety wherein a nucleobase or amodified nucleobase is covalently linked to a sugar or modified sugar,and the sugar or modified sugar is covalently linked to a phosphategroup or a modified phosphorous-atom moiety.

The term “chiral reagent” refers to a compound that is chiral orenantiopure and can be used for asymmetric induction in nucleic acidsynthesis.

The term “chiral ligand” or “chiral auxiliary” refers to a moiety thatis chiral or enantiopure and controls the stereochemical outcome of areaction.

In a condensation reaction, the term “condensing reagent” refers to areagent that activates a less reactive site and renders it moresusceptible to attack by a nucleophile.

The term “blocking moiety” refers to a group that transiently masks thereactivity of a functional group. The functional group can besubsequently unmasked by removal of the blocking moiety.

The terms “boronating agents”, “sulfur electrophiles”, “seleniumelectrophiles” refer to compounds that are useful in the modifying stepused to introduce BH₃, S, and Se groups, respectively, for modificationat the phosphorus atom.

The term “moiety” refers to a specific segment or functional group of amolecule. Chemical moieties are often recognized chemical entitiesembedded in or appended to a molecule.

The term “solid support” refers to any support which enables syntheticmass production of nucleic acids and can be reutilized at need. As usedherein, the term refers to a polymer that is insoluble in the mediaemployed in the reaction steps performed to synthesize nucleic acids,and is derivatized to comprise reactive groups.

The term “linking moiety” refers to any moiety optionally positionedbetween the terminal nucleoside and the solid support or between theterminal nucleoside and another nucleoside, nucleotide, or nucleic acid.

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” are used interchangeably herein. These terms refers to anapproach for obtaining beneficial or desired results including but notlimited to therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant eradication or amelioration of theunderlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the patient, notwithstanding that thepatient may still be afflicted with the underlying disorder. Forprophylactic benefit, the compositions may be administered to a patientat risk of developing a particular disease, or to a patient reportingone or more of the physiological symptoms of a disease, even though adiagnosis of this disease may not have been made.

A “therapeutic effect,” as that term is used herein, encompasses atherapeutic benefit and/or a prophylactic benefit as described above. Aprophylactic effect includes delaying or eliminating the appearance of adisease or condition, delaying or eliminating the onset of symptoms of adisease or condition, slowing, halting, or reversing the progression ofa disease or condition, or any combination thereof.

An “alkyl” group refers to an aliphatic hydrocarbon group. The alkylmoiety may be a saturated alkyl group (which means that it does notcontain any units of unsaturation, e.g. carbon-carbon double bonds orcarbon-carbon triple bonds) or the alkyl moiety may be an unsaturatedalkyl group (which means that it contains at least one unit ofunsaturation). The alkyl moiety, whether saturated or unsaturated, maybe branched, straight chain, or include a cyclic portion. The point ofattachment of an alkyl is at a carbon atom that is not part of a ring.

The “alkyl” moiety may have 1 to 10 carbon atoms (whenever it appearsherein, a numerical range such as “1 to 10” refers to each integer inthe given range; e.g., “1 to 10 carbon atoms” means that the alkyl groupmay consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., upto and including 10 carbon atoms, although the present definition alsocovers the occurrence of the term “alkyl” where no numerical range isdesignated). Alkyl includes both branched and straight chain alkylgroups. The alkyl group of the compounds described herein may bedesignated as “C₁-C₆ alkyl” or similar designations. By way of exampleonly, “C₁-C₆ alkyl” indicates that there are one, two, three, four,five, or six carbon atoms in the alkyl chain, i.e., the alkyl chain isselected from the group consisting of methyl, ethyl, propyl, iso-propyl,n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyl groupsinclude, but are in no way limited to, methyl, ethyl, propyl, isopropyl,butyl, isobutyl, tertiary butyl, pentyl, hexyl, allyl,cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl,cyclohexylmethyl, and the like. In one aspect, an alkyl is a C₁-C₆alkyl.

As used herein, the term “aryl” refers to an aromatic ring wherein eachof the atoms forming the ring is a carbon atom. Aryl rings are formed byfive, six, seven, eight, nine, or more than nine carbon atoms. Arylgroups are a substituted or unsubstituted. In one aspect, an aryl is aphenyl or a naphthalenyl. Depending on the structure, an aryl group canbe a monoradical or a diradical (i.e., an arylene group). In one aspect,an aryl is a C₆-C₁₀aryl.

“Heteroaryl” or alternatively, “heteroaromatic” refers to a 5- to18-membered aromatic radical (e.g., C₅-C₁₃ heteroaryl) that includes oneor more ring heteroatoms selected from nitrogen, oxygen and sulfur, andwhich may be a monocyclic, bicyclic, tricyclic or tetracyclic ringsystem. Whenever it appears herein, a numerical range such as “5 to 18”refers to each integer in the given range; e.g., “5 to 18 ring atoms”means that the heteroaryl group may consist of 5 ring atoms, 6 ringatoms, etc., up to and including 18 ring atoms. An N-containing“heteroaromatic” or “heteroaryl” moiety refers to an aromatic group inwhich at least one of the skeletal atoms of the ring is a nitrogen atom.The polycyclic heteroaryl group may be fused or non-fused. Theheteroatom(s) in the heteroaryl radical is optionally oxidized. One ormore nitrogen atoms, if present, are optionally quaternized. Theheteroaryl is attached to the rest of the molecule through any atom ofthe ring(s). Examples of heteroaryls include, but are not limited to,azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxolyl,benzofuranyl, benzooxazolyl, benzo[d]thiazolyl, benzothiadiazolyl,benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl,benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl,benzoxazolyl, benzopyranyl, benzopyranonyl, benzofuranyl,benzofuranonyl, benzofurazanyl, benzothiazolyl, benzothienyl(benzothiophenyl), benzothieno[3,2-d]pyrimidinyl, benzotriazolyl,benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl,cyclopenta[d]pyrimidinyl,6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl,5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl,6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl,dibenzothiophenyl, furanyl, furazanyl, furanonyl, furo[3,2-c]pyridinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyridinyl,isothiazolyl, imidazolyl,indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl,isoquinolyl, indolizinyl, isoxazolyl,5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl,1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl,5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1-phenyl-1H-pyrrolyl,phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl,purinyl, pyranyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl,pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl,pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl,quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl,5,6,7,8-tetrahydroquinazolinyl,5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl,6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl,5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl,thiapyranyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl,thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pridinyl, and thiophenyl (i.e.thienyl). Unless stated otherwise specifically in the specification, aheteraryl moiety is optionally substituted by one or more substituentswhich are independently: alkyl, heteroalkyl, alkenyl, alkynyl,cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, hydroxy, halo, cyano, nitro, oxo, thioxo,trimethylsilanyl, —OR^(a), —SR^(a), —OC(O)R^(a), —N(R^(a))₂, —C(O)R^(a),—C(O)OR^(a), —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a),—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), or —S(O)_(t)N(R^(a))₂ (where t is 1 or 2) where each R^(a) isindependently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl,heteroaryl or heteroarylalkyl.

The term “alicyclic” refers to an all carbon moiety that is bothaliphatic and cyclic. Alicyclic groups contain one or more all-carbonrings which may be either saturated or unsaturated, but do not havearomatic character. Alicyclic groups are substituted or unsubstitutedand may contain from one to ten carbon atoms. In one aspect, analicyclic is a monocyclic cycloalkane. In another aspect an alicyclic isa bicyclic cycloalkane.

The term “aralkyl” refers to an alkyl group substituted with an arylgroup. Suitable aralkyl groups include benzyl, picolyl, and the like,all of which may be optionally substituted.

An “acyl moiety” refers to an alkyl(C═O), aryl(C═O), or aralkyl(C═O)group. An acyl moiety can have an intervening moiety (Y) that is oxy,amino, thio, or seleno between the carbonyl and the hydrocarbon group.For example, an acyl group can be alkyl-Y—(C═O), aryl-Y—(C═O) oraralkyl-Y—(C═O).

“Alkenyl” groups are straight chain, branch chain, and cyclichydrocarbon groups containing at least one carbon-carbon double bond.Alkenyl groups can be substituted.

“Alkynyl” groups are straight chain, branch chain, and cyclichydrocarbon groups containing at least one carbon-carbon triple bond.Alkynyl groups can be substituted.

An “alkoxy” group refers to an alklyl group linked to oxygen i.e.(alkyl)-O-group, where alkyl is as defined herein. Examples includemethoxy (—OCH₃) or ethoxy (—OCH₂CH₃) groups.

An “alkenyloxy” group refers to an alkenyl group linked to oxygen i.e.(alkenyl)-O-group, where alkenyl is as defined herein.

An “alkynyloxy” group refers to an alkynyl group linked to oxygen i.e.(alkynyl)-O-group, where alkynyl is as defined herein.

An “aryloxy” group refers to an aryl group linked to oxygen i.e.(aryl)-O-group, where the aryl is as defined herein. An example includesphenoxy (—OC₆H₅).

The term “alkylseleno” refers to an alkyl group having a substitutedseleno group attached thereto i.e. (alkyl)-Se-group, wherein alkyl isdefined herein.

The term “alkenylseleno” refers to an alkenyl group having a substitutedseleno group attached thereto i.e. (alkenyl)-Se-group, wherein alkenylis defined herein.

The term “alkynylseleno” refers to an alkynyl group having a substitutedseleno group attached thereto i.e. (alkynyl)-Se-group, wherein alkenylis defined herein.

The term “alkylthio” refers to an alkyl group attached to a bridgingsulfur atom i.e. (alkyl)-S-group, wherein alkyl is defined herein. Forexample, an alkylthio is a methylthio and the like.

The term “alkenylthio” refers to an alkenyl group attached to a bridgingsulfur atom i.e. (alkenyl)-S-group, wherein alkenyl is defined herein.

The term “alkynylthio” refers to an alkynyl group attached to a bridgingsulfur atom i.e. (alkynyl)-S-group, wherein alkenyl is defined herein.

The term “alkylamino” refers to an amino group substituted with at leastone alkyl group i.e. —NH(alkyl) or —N-(alkyl)₂, wherein alkyl is definedherein.

The term “alkenylamino” refers to an amino group substituted with atleast one alkenyl group i.e. —NH(alkenyl) or —N-(alkenyl)₂, whereinalkenyl is defined herein.

The term “alkynylamino” refers to an amino group substituted with atleast one alkynyl group i.e. —NH(alkynyl) or —N-(alkynyl)₂, whereinalkynyl is defined herein.

The term “halogen” is intended to include fluorine, chlorine, bromineand iodine.

A “fluorescent group” refers to a molecule that, when excited with lighthaving a selected wavelength, emits light of a different wavelength.Fluorescent groups include, but are not limited to, indole groups,fluorescein, tetramethylrhodamine, Texas Red, BODIPY,5-[(2-aminoethyl)amino]napthalene-1-sulfonic acid (EDANS), coumarin andLucifer yellow.

An “ammonium ion” is a positively charged polyatomic cation of thechemical formula NH₄ ⁺.

An “alkylammonium ion” is an ammonium ion that has at least one of itshydrogen atoms replaced by an alkyl group, wherein alkyl is definedherein. Examples include triethylammonium ion,N,N-diisopropylethylammonium ion.

An “iminium ion” has the general structure R₂C═NR₂ ⁺. The R groups referto alkyl, alkenyl, alkynyl, aryl groups as defined herein. A“heteroaromatic iminium ion” refers to an imminium ion where thenitrogen and its attached R groups form a heteroaromatic ring. A“heterocyclic iminium ion” refers to an imminium ion where the nitrogenand its attached R groups form a heterocyclic ring.

The terms “amino” or “amine” refers to a —N(R^(h))₂ radical group, whereeach R^(h) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl,heteroaryl or heteroarylalkyl, unless stated otherwise specifically inthe specification. When a —N(R^(f))₂ group has two R^(f) other thanhydrogen they can be combined with the nitrogen atom to form a 4-, 5-,6-, or 7-membered ring. For example, —N(R^(f))₂ is meant to include, butnot be limited to, 1-pyrrolidinyl and 4-morpholinyl. Any one or more ofthe hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl,aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkylare optionally substituted by one or more substituents whichindependently are alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy,halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl,—OR^(i), —SR^(i), —OC(O)R^(i), —N(R^(i))₂, —C(O)R^(i), —C(O)OR^(i),—OC(O)N(R^(i))₂, —C(O)N(R^(i))₂, —N(R^(i))C(O)OR^(i),—N(R^(i))C(O)R^(i), —N(R^(i))C(O)N(R^(i))₂, N(R^(i))C(NR^(i))N(R^(i))₂,—N(R^(i))S(O)_(t)R^(i) (where t is 1 or 2), —S(O)_(t)OR^(f) (where t is1 or 2), or —S(O)_(t)N(R^(f))₂ (where t is 1 or 2), where each R^(i) isindependently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl,heteroaryl or heteroarylalkyl.

“Carbamate” as used herein, refers to a moiety attached to an aminogroup which has the formula —C(O)OR where R is alkyl, fluoroalkyl,carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocyclyl,heterocyclylalkyl, heteroaryl or heteroarylalkyl. Examples include butare not limited to Boc (tert-butyl—OC(O)—), CBz (benzyl—OC(O)—), Teoc(Me₃SiCH₂CH₂OC(O)—), alloc (allyl—OC(O)—), or Fmoc(9-fluorenylmethyl-OC(O)—).

“Substituted silyl” as used herein, refers to a moiety which has theformula R₃Si—. Examples include, but are not limited to, TBDMS(tert-butyldimethylsilyl), TBDPS (tert-butyldiphenylsilyl) or TMS(trimethylsilyl).

The term “thiol” refers to —SH groups, and include substituted thiolgroups i.e. —SR^(j) groups, wherein R^(j) are each independently asubstituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, arylaralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.

Methods of Synthesis General Discussion of the Methods of Synthesis of aNucleic Acid Comprising a Chiral X-Phosphonate Moiety.

The present method provides for an efficient synthesis of phosphorusatom-modified nucleic acids wherein the stereochemical configuration ata phosphorus atom is controlled, thus producing a stereodefinedoligonucleotide. The method eliminates the need for complex separationsof diastereomeric mixtures and allows for the use of readily availableinexpensive achiral starting materials. The method of synthesisdisclosed herein comprises an asymmetric reaction of an achiralH-phosphonate moiety (Formula 2) with a nucleoside comprising anucleophilic moiety, such as a hydroxy group, (Formula 4-1, where Q₁ isany of a blocking group, a linking moiety to a support or to anucleotide chain) to provide a phosphorous atom-modified nucleic acidcomprising a chiral X-phosphonate moiety, which is a compound of Formula1, as shown in Scheme 1. In such manner, a nucleotide polymer oroligomer having high diastereomeric purity is produced. In someembodiments, the nucleic acid contains modifications at the nucleobases,sugar moiety, and/or protective groups.

The reaction of a molecule comprising an achiral H-phosphonate moiety ofFormula 2 with a nucleoside comprising nucleophilic moiety of Formula4-1 results in the formation of a condensed intermediate; which isconverted to the nucleic acid comprising a chiral X-phosphonate moiety.The synthesis of the condensed intermediate comprises the steps of (a)activation of the compound of Formula 2 with a condensing agent, (b)reaction with a chiral reagent, followed by (c) reaction with thecompound of Formula 4-1. The general scheme is shown in Scheme 2. Thechiral reagent becomes attached to the condensed intermediate as achiral auxiliary group. In the process provided herein, the steps(a)-(c) leading to the condensed intermediate may be performed withoutisolating any intermediates, i.e., in the same pot or in one-pot. Thus,the process obviates the need for isolation of discrete intermediates.The process disclosed herein can be performed in solution or on solidsupport. Depending on the reaction conditions, addition of an activatingreagent may be useful for the condensation step. For example, theactivating reagent can be added to the reaction after steps (a)-(c) havebeen completed or can be added to the reaction at the same time as steps(a)-(c).

In an embodiment the condensed intermediate is converted to a nucleicacid comprising a chiral X phosphonate moiety of Formula 1 by cappingthe chiral auxiliary on the condensed intermediate with a moiety A,which is an acyl, aryl, alkyl, aralkyl, or silyl moiety, and modifyingthe phosphorus to introduce J, which is S, Se, or BH₃, producing acompound of Formula 5-1. In one embodiment (Option A, Scheme 3), thecompound of Formula 5-1 is converted to the compound of Formula 1, whereX is S, Se, or BH₃, and n is 1 (dimer), by cleaving the chiralauxiliary, and deblocking blocking groups and cleaving from solidsupport if desired. When forming the dimer, the capping step in Scheme 3is optional. Alternatively (Option B, Scheme 3), the compound of Formula5-1 is subjected to chain elongation by repeating the steps to produce acondensed intermediate where a further monomer of Formula 2 is added tothe oligonucleotide. The steps of capping, modifying, deblocking, andchain elongation are repeated until the desired n is achieved. At thatpoint, the chiral auxilliaries at each phosphonate are cleaved, theremaining blocking groups are cleaved, including cleaving from a solidsupport, if desired, to produce the compound of Formula 1, where X is S,Se, or BH₃, and n is greater than or equal to 2 and less than about 200.

In another method provided herein, the condensed intermediate isconverted to a nucleic acid comprising a chiral X phosphonate moiety ofFormula 1 by acidifying the condensed intermediate to remove theblocking group at R¹, which also removes the chiral auxiliary. In oneembodiment (Option A, Scheme 4), the compound of Formula 4 is modifiedto introduce an X moiety at phosphorus, to produce a compound of Formula1, which is deblocked to remove remaining blocking groups, and removefrom a synthesis support, if desired, to produce a compound of Formula 1wherein R³ is hydrogen and n is 1.

Alternatively, the compound of Formula 4 in Scheme 4 (Option B) issubjected to the step of chain elongation reaction, and then acidifiedto deblock the R¹ blocking group of the newly added nucleoside. Thechain elongation step and R¹ deblocking step are performed for mrepetitions. At that point, the compound of Formula 4, wherein m isequal to n−1, is modified to introduce an X moiety at each phosphorus,to produce a compound of Formula 1, which is deblocked to removeremaining blocking groups, and remove from a synthesis support, ifdesired, to produce a compound of Formula 1 wherein R³ is hydrogen and nis greater than or equal to 2 and less than about 200.

In both Option A and Option B of Scheme 4, X is alkyl, alkoxy, aryl,alkylthio, acyl, —NR^(f)R^(f), alkenyloxy, alkynyloxy, alkenylthio,alkynylthio, —S⁻Z⁺, —Se⁻Z⁺, or —BH₃ ⁻Z⁺, where each R^(f) isindependently hydrogen, alkyl, alkenyl, alkynyl, or aryl; Z⁺ is ammoniumion, alkylammonium ion, heteroaromatic iminium ion, or heterocycliciminium ion, any of which is primary, secondary, tertiary or quaternary,or Z is a monovalent metal ion. In other embodiments, Z is pyridiniumion, triethylammonium ion, N,N-diisopropylethylammonium ion,1,8-diazabicyclo[5.4.0]undec-7-enium ion, sodium ion, or potassium ion.

Phosphorus Atom Modified Nucleic Acid Comprising a Chiral X-PhosphonateMoiety of Formula 1.

The process of the invention provides a nucleic acid comprising a chiralX-phosphonate moiety of the following general Formula 1-1 or Formula1-2:

Wherein the X-phosphonate moiety connects natural nucleoside moieties orunnatural nucleoside moieties wherein the natural ribose ring isreplaced by a larger or smaller oxygen containing ring or wherein thering is replaced by a noncyclic structure wherein W₃ is —S—, —O—,substituted or unsubstituted amino, alkylene, alkenylene, or alkynylene.In other embodiments of the nucleic acid, the X-phosphonate moietyconnects natural nucleoside moieties with unnatural nucleoside moieties.In yet other embodiments of the nucleic acid, the X-phosphonate moietyconnects nucleoside moieties with different sugar moieties to oneanother.

In one embodiment of the invention, the nucleic acid comprising a chiralX-phosphonate moiety is a compound of Formula 1:

In Formula 1, R¹ is —OH, —SH, —NR^(d)R^(d), —N₃, halogen, hydrogen,alkyl, alkenyl, alkynyl, alkyl-Y¹—, alkenyl-Y¹—, alkynyl-Y¹—, aryl-Y¹—,heteroaryl-Y¹—, —P(O)(R^(e))₂, —HP(O)(R^(e)), —OR^(a) or —SR^(c).

Y¹ is O, NR^(d), S, or Se.

R^(a) is a blocking moiety.

R^(c) is a blocking group.

Each instance of R^(d) is independently hydrogen, alkyl, alkenyl,alkynyl, aryl, acyl, substituted silyl, carbamate, —P(O)(R^(e))₂, or—HP(O)(R^(e)).

Each instance of R^(e) is independently hydrogen, alkyl, aryl, alkenyl,alkynyl, alkyl-Y²—, alkenyl-Y²—, alkynyl-Y²—, aryl-Y²—, orheteroaryl-Y²—, or a cation which is Na⁺¹, Li⁺¹, or K⁺¹.

Y² is O, NR^(d), or S.

Each instance of R² is independently hydrogen, —OH, —SH, —NR^(d)R^(d),—N₃, halogen, alkyl, alkenyl, alkynyl, alkyl-Y¹—, alkenyl-Y¹—,alkynyl-Y¹—, aryl-Y¹—, heteroaryl-Y¹—, —OR^(b), or —SR^(c), whereinR^(b) is a blocking moiety.

Each instance of Ba is independently a blocked or unblocked adenine,cytosine, guanine, thymine, uracil or modified nucleobase.

Each instance of X is independently alkyl, alkoxy, aryl, alkylthio,acyl, —NR^(f)R^(f), alkenyloxy, alkynyloxy, alkenylthio, alkynylthio,—S⁻Z⁺, —Se⁻Z⁺, or —BH₃ ⁻Z⁺.

Each instance of R^(f) is independently hydrogen, alkyl, alkenyl,alkynyl, or aryl.

Z⁺ is ammonium ion, alkylammonium ion, heteroaromatic iminium ion, orheterocyclic iminium ion, any of which is primary, secondary, tertiaryor quaternary, or Z is a monovalent metal ion.

R³ is hydrogen, a blocking group, a linking moiety connected to a solidsupport or a linking moiety connected to a nucleic acid; and n is aninteger of 1 to about 200.

In an embodiment, any of the R² groups is substituted by, for example, afluorescent moiety, a biotin moiety, or an avidin moiety.

In one embodiment, the nucleic acid described herein is prepared fromall ribonucleotide monomers. In another embodiment, it is prepared fromall deoxyribonucleotide monomers. In yet another embodiment, the nucleicacid is prepared from a mixture of ribonucleotide or deoxyribonucleotidemonomers. In one embodiment the nucleic acid is a mixture of RNA and DNAmoieties. In another embodiment, the nucleic acid comprises asubstituent at R² which is not found in RNA or DNA nucleic acids.

Ba represents a nucleobase, which is a natural or modified nucleobase.Each instance of the nucleobase is independently blocked or unblocked.

Each instance of X is independently alkyl, alkoxy, aryl, alkylthio,acyl, —NR^(f)R^(f), alkenyloxy, alkynyloxy, alkenylthio, alkynylthio,—S⁻Z⁺, —Se⁻Z⁺, or —BH₃ ⁻Z⁺, wherein each instance of R^(f) isindependently hydrogen, alkyl, alkenyl, alkynyl, or aryl; Z⁺ is ammoniumion, alkylammonium ion, heteroaromatic iminium ion, or heterocycliciminium ion, any of which is primary, secondary, tertiary or quaternary,or Z is a monovalent metal ion. In some embodiments, X is alkyl, alkoxy,—NR^(f)R^(f), —S⁻Z⁺, or —BH₃ ⁻Z⁺. In other embodiments, Z is pyridiniumion, triethylammonium ion, N,N-diisopropylethylammonium ion,1,8-diazabicyclo[5.4.0]undec-7-enium ion, sodium ion, or potassium ion.

R³ is hydrogen, a blocking group, a linking moiety connected to a solidsupport or a linking moiety connected to a nucleic acid, which areprepared using methods herein or known in the art. The nucleic acidattached to R³ that are prepared using any known method comprisephosphorus atoms that are modified, unmodified, or mixtures of modifiedand unmodified phosphorus and comprise any configuration at thephosphorus atom. In one embodiment, R³ is a linking moiety attached toanother nucleoside or nucleotide.

X-Phosphonate Moiety.

As used herein, X-phosphonate moiety refers to the phosphorus atom ofthe internucleoside backbone linkage that is modified to be covalentlybonded to a moiety X, where X can be, but not limited to, sulphur,selenium, alkyl, boron, acyl, amino, thiol, or alkoxy. The X moietymodifies the phosphorus atom by replacement of one of the oxygen atomsin the internucleoside backbone. The internucleoside backbone linkagesare shown below (within the dashed rectangular boxes) for two nucleicacid fragments as non-limiting examples. The left hand structure belowshows the phosphate group found in natural internucleoside backbonelinkages. The right hand structure below shows a X-phosphonate moiety asthe internucleoside backbone linkage.

A phosphorothioate moiety comprises a sulphur moiety as the X moiety. Aphosphoroselenoate moiety comprises a selenium moiety as the X moiety.An alkyl phosphonate moiety (e.g. methyl phosphonate) comprises an alkylgroup (e.g. methyl group) as the X moiety. A boronophosphonate moietycomprises a borane group as the X moiety.

In an embodiment, the nucleic acid comprises phosphorothioate groups inthe backbone linkages. In some embodiments, the nucleic acid comprisesphosphoroselenoate groups in the backbone linkages. In otherembodiments, the nucleic acid comprises alkyl phosphonate groups (e.g.methyl phosphonate) in the backbone linkages. In yet other embodiments,the nucleic acid comprise boronophosphonate groups in the backbonelinkages.

Each X moiety can be independently chosen from the various X moietiesdescribed herein. This allows multiple X moieties to be present withinone nucleic acid. In one embodiment, the same X moiety is usedthroughout the nucleic acid. In other embodiments, different X moietiesare used throughout the nucleic acid. For example, within one nucleicacid, some of the X-phosphonates are phosphothioate moieties while otherX-phosphonates within the same nucleic acid are alkyl phosphonatemoieties. It will be evident to one skilled in the art that othervariations and alternations of phosphorus modifications are possible anddepend on the use and applications of these nucleic acids. In someembodiments, the choice for the X moiety depends on the biochemicalproperties of the nucleic acid and its interactions with biologicalsamples.

Configuration of X-Phosphonate Moiety.

The methods described herein are useful for controlling theconfiguration of each phosphorus atom in the internucleoside backbonelinkage. The chiral reagent permits the specific control of thechirality at the X-phosphonate. Thus, either a R_(P) or S_(P)configuration can be selected in each synthesis cycle, permittingcontrol of the overall three dimensional structure of the nucleic acidproduct. In some embodiments, the selection of R_(P) and S_(P)configurations is made to confer a specific three dimensionalsuperstructure to the nucleic acid chain.

In some embodiments, each X-phosphonate moiety can have a R_(P)configuration. In other embodiments, each X-phosphonate moiety can havea S_(P) configuration. In another embodiment, each X-phosphonate moietyindependently can have a R_(P) configuration or a S_(P) configuration.In specific embodiments, the X-phosphonate moieties alternate betweenR_(P) and S_(P) such as R_(P), S_(P), R_(P) or S_(P), R_(P), S_(P)throughout the nucleic acid. In other specific embodiments, theX-phosphonate moieties contain repeated configurations of R_(P), R_(P),S_(P), S_(P) throughout the nucleic acid. In yet other embodiments, thenucleic acid comprises all R_(P) configurations. In further embodiments,the nucleic acid comprises all S_(P) moieties. In some embodiments, the5′ and 3′ terminal internucleoside backbone linkages are of the S_(P)configuration and the internal internucleoside backbone linkages are allof the R_(P) configuration. The embodiments described herein serve asexamples of how the configuration can be controlled using these methods.The nucleic acid described herein is not limited to these configurationpatterns. It will be evident to one skilled in the art that othervariations and alternations in the R_(P) and S_(P) configurations arepossible and depend on the use and applications of the nucleic acid.

Purity Determination of X-Phosphonate Configurations.

The purity of the configuration at each X-phosphonate moiety in thenucleic acid is determined using conventional analytical methods suchas, but not limited to, ³¹P NMR spectroscopy or reverse-phase HPLC.Using methods described herein, in an embodiment, each X-phosphonatemoiety of the compound can be more than 80% diastereomerically pure. Inan embodiment, each X-phosphonate moiety of the compound can be morethan 60% diastereomerically pure. In an embodiment, each X-phosphonatemoiety of the compound can be more than 70% diastereomerically pure. Inan embodiment, each X-phosphonate moiety of the compound can be morethan 85% diastereomerically pure. In an embodiment, each X-phosphonatemoiety of the compound can be more than 90% diastereomerically pure. Inan embodiment, each X-phosphonate moiety of the compound can be morethan 95% diastereomerically pure. In another embodiment, eachX-phosphonate moiety of the compound can be more than 98%diastereomerically pure. In another embodiment, each X-phosphonatemoiety of the compound can be more than 99% diastereomerically pure. Inan embodiment, each X-phosphonate moiety of the compound can be morethan about 60%, more than about 70%, more than about 80%, more thanabout 83%, more than about 84%, more than about 85%, more than about86%, more than about 87%, more than about 88%, more than about 89%, morethan about 90%, more than about 91%, more than about 92%, more thanabout 93%, more than about 94%, more than about 95%, more than about96%, more than about 97%, more than about 98%, or more than about 99%diastereomerically pure. In one embodiment, each X-phosphonate moietycan be from about 60% to about 99.9% diastereomerically pure. In oneembodiment, each X-phosphonate moiety can be from about 60% to about 99%diastereomerically pure. In one embodiment, each X-phosphonate moietycan be from about 60% to about 70% diastereomerically pure. In oneembodiment, each X-phosphonate moiety can be from about 70% to about 80%diastereomerically pure. In one embodiment, each X-phosphonate moietycan be from about 80% to about 90% diastereomerically pure. In oneembodiment, each X-phosphonate moiety can be from about 80% to about 99%diastereomerically pure. In one embodiment, each X-phosphonate moietycan be from about 85% to about 95% diastereomerically pure. In oneembodiment, each X-phosphonate moiety can be from about 90% to about 95%diastereomerically pure. In one embodiment, each X-phosphonate moietycan be from about 95% to about 99% diastereomerically pure. In oneembodiment, each X-phosphonate moiety can be from about 90% to about99.9% diastereomerically pure.

The amount of a particular configuration over another configurationaffects the three-dimensional structure of the nucleic acids as well astheir stability. Accordingly, different configurations affect thebiological, chemical, and physical properties of the nucleic acids. Inone embodiment, the nucleic acid comprises a greater percentage of S_(P)configuration than R_(P) configuration. In another embodiment, thenucleic acid comprises a greater percentage of R_(P) configuration thanS_(P) configuration. In another embodiment, the nucleic acid comprisesthe same percentage of R_(P) configuration as S_(P) configuration. Inone embodiment, the nucleic acid can comprise 0-20% R_(P) configuration.In one embodiment, the nucleic acid can comprise 20-40% R_(P)configuration. In one embodiment, the nucleic acid can comprise 40-60%R_(P) configuration. In one embodiment, the nucleic acid can comprise60-80% R_(P) configuration. In one embodiment, the nucleic acid cancomprise 80-100% R_(P) configuration. In one embodiment, the nucleicacid can comprise 0-20% S_(P) configuration. In one embodiment, thenucleic acid can comprise 20-40% S_(P) configuration. In one embodiment,the nucleic acid can comprise 40-60% S_(P) configuration. In oneembodiment, the nucleic acid can comprise 60-80% S_(P) configuration. Inone embodiment, the nucleic acid can comprise 80-100% S_(P)configuration.

Length of the Phosphorus Atom Modified Nucleic Acid.

The nucleic acid comprising a chiral X-phosphonate moiety of Formula 1comprises from about 1 nucleoside to about 200 nucleosides. In someembodiments, the nucleic acid comprising a chiral X-phosphonate moietyof Formula 1 are further combined into oligomers or polymers. In someembodiments, the nucleic acid of Formula 1 is a dimer. In otherembodiments, the nucleic acid of Formula 1 comprises up to about 100nucleosides. In other embodiments, the nucleic acid of Formula 1comprises up to about 150 nucleosides. In other embodiments, the nucleicacid of Formula 1 comprises up to about 200 nucleosides. In otherembodiments, the nucleic acid of Formula 1 comprises up to about 300nucleosides. In some embodiments, the nucleic acid of Formula 1comprises from 1 to about 200 nucleosides. In other embodiments, thenucleic acid comprises from 1 to about 150 nucleosides. In furtherembodiments, nucleic acid contains from 1 to about 10 nucleosides. Inother embodiments, the nucleic acid contains from about 10 to about 50nucleosides. In further embodiments, nucleic acid contains from about 10to about 100 nucleosides. In some embodiments, the nucleic acidcomprises from 1 to about 5 nucleosides, or about 5 to about 10nucleosides, or about 5 to about 15 nucleosides, or about 10 to about 20nucleosides, or about 15 to about 25 nucleosides, or about 20 to about30 nucleosides, or about 25 to about 35 nucleosides, or about 30 toabout 40 nucleosides. In some embodiments of Formula 1, n is an integerof 1 to about 200. In some embodiments of Formula 1, n is an integer of1 to about 150. In some embodiments of Formula 1, n is an integer of 1to about 10. In some embodiments of Formula 1, n is an integer of 10 toabout 50. In some embodiments of Formula 1, n is an integer of 10 toabout 100. In some embodiments of Formula 1, n is an integer of 1 toabout 5, or about 5 to about 10, or about 5 to about 15, or about 10 toabout 20, or about 15 to about 25, or about 20 to about 30, or about 25to about 35, or about 30 to about 40.

Additional Variations of Phosphorus Atom Modified Nucleic Acid.

The nucleic acid of Formula 1 can be single-stranded. In someembodiments, the nucleic acid of Formula 1 is hybridized to acomplementary strand to form a double-stranded nucleic acid.

In some embodiments, the nucleic acid of Formula 1 can comprise an openlinear structure. In other embodiments, the respective ends of thenucleic acid of Formula 1 are joined to form a circular structure.

Within a nucleic acid, the sugar component of each unit comprises thesame or different sugars. In some embodiments, the sugars are modifiedsugars or sugars that are substituted. In some embodiments, the sugarsare all ribose sugar moieties. In some embodiments, the sugars are alldeoxyribose sugar moieties. In other embodiments, the sugars are allpentofuranose, pentopyranose, or hexopyranose moieties. In furtherembodiments, the sugar component comprises closed ring structures oropen structures.

Within the nucleic acid structure, the phosphorous atom bridges arecommonly referred to as forming the internucleoside backbone of thenucleic acids. The internucleoside backbone linkages in nucleic acidsinclude, and are not limited to, 2′ to 5′ phosphorous atom bridges, 3′to 5′ phosphorous atom bridges, 5′ to 3′ phosphorous atom bridges, andthe 3′ to 2′ phosphorous atom bridges and 4′ to 2′ bridges described inU.S. Pat. No. 6,608,186 and Joyce, G. F. Nature, 2002, 418, 214-220.Non-limiting examples of these variations in internucleoside backbonelinkages are shown below:

Depending on the sugar or modified sugar component, other types ofphosphorous atom bridges are also contemplated including, and notlimited to, methylene bisphosphonate bridges shown below and describedin Xu, L. et al, J. Med. Chem., 2005, 48, 4177-4181.

The nucleic acid of Formula 1 can comprise the same or differentnucleobases. In some embodiments, the nucleic acid of Formula 1comprises all the same nucleobases. In other embodiments, the nucleicacid of Formula 1 comprises all different nucleobases. In otherembodiments, the nucleic acid of Formula 1 comprises the naturallyoccurring nucleobases. In some embodiments, the nucleic acid of Formula1 comprises modified nucleobases. In yet other embodiments, the nucleicacid contain nucleobases that mimic the nucleobase sequence of a nucleicacid found in nature. In some embodiments, the nucleic acid of Formula 1comprises a mixture of naturally occurring nucleobases and modifiednucleobases.

Molecules Comprising an Achiral H-Phosphonate Moiety.

The molecule comprising an achiral H-phosphonate moiety is a compound ofFormula 2:

In Formula 2, R¹ is —NR^(d)R^(d), —N₃, halogen, hydrogen, alkyl,alkenyl, alkynyl, alkyl-Y¹—, alkenyl-Y¹—, alkynyl-Y¹—, aryl-Y¹—,heteroaryl-Y¹—, —P(O)(R^(e))₂, —HP(O)(R^(e)), —OR^(a), or —SR^(c).

Y¹ is O, NR^(d), S, or Se.

R^(a) is a blocking moiety.

R^(c) is a blocking group.

Each instance of R^(d) is independently hydrogen, alkyl, alkenyl,alkynyl, aryl, acyl, substituted silyl, carbamate, —P(O)(R^(e))₂, or—HP(O)(R^(e)).

Each instance of R^(e) is independently alkyl, aryl, alkenyl, alkynyl,alkyl-Y²—, alkenyl-Y²—, alkynyl-Y²—, aryl-Y²—, or heteroaryl-Y²—.

Y² is O, NR^(d), or S.

R² is hydrogen, —NR^(d)R^(d), N₃, halogen, alkyl, alkenyl, alkynyl,alkyl-Y¹—, alkenyl-Y¹—, alkynyl-Y¹—, aryl-Y¹—, heteroaryl-Y¹—, —OR^(b),or —SR^(c), wherein R^(b) is a blocking moiety.

Ba is a blocked or unblocked adenine, cytosine, guanine, thymine, uracilor modified nucleobase.

Z⁺ is ammonium ion, alkylammonium ion, heteroaromatic iminium ion, orheterocyclic iminium ion, any of which is primary, secondary, tertiaryor quaternary, or a monovalent metal ion.

In some embodiments, Z is pyridinium ion, triethylammonium ion,N,N-diisopropylethylammonium ion, 1,8-diazabicyclo[5.4.0]undec-7-eniumion, sodium ion, or potassium ion.

In some embodiments, the sugar is a ribose ring. In other embodiments,the sugar is deoxyribose, pentofuranose, pentopyranose, or hexopyranosemoieties. In other embodiments, the sugar is a modified sugar. In someembodiments, the sugar is a glycerol analogue or a sugar withsubstitutions.

The H-phosphonate nucleoside monomers are easily prepared and stable.Methods of their preparation have been described (see e.g. Froehler, B.C. Methods in Molecular Biology. In Protocols for Oligonucleotides andAnalogs; Agrawal, S., Ed.; Humana: Totowa, 1993; vol 20, p 63-80).

In some embodiments, the nucleoside monomer comprises an achiralH-phosphonate moiety attached to the nucleoside at the 3′ position. Inyet further embodiments, nucleoside monomer comprises an achiralH-phosphonate moiety attached to the nucleoside moiety at the 3′position through an intervening linking moiety. In specific embodiments,the intervening linking moiety is a methylene group (see. e.g.WO/2001/02415). In some embodiments the H-phosphonate moiety is attachedto the 2′ position of the nucleoside monomer. In other embodiments, thenucleoside monomer comprises an achiral H-phosphonate moiety attached tothe nucleoside at the 5′ position.

Compounds with a Free Nucleophilic Moiety.

The compound comprising a free nucleophilic moiety is a compound ofFormula 4-1 and reacts at the phosphorus center of the chiralintermediate. The direction of attack at phosphorus by the nucleophilicgroup or moiety depends on the substituents of the chiral auxiliary onthe chiral intermediate (condensed intermediate). In an embodiment,addition of an activating reagent can be useful for helping the compoundcomprising a free nucleophilic moiety to react at the phosphorus centerof the chiral intermediate.

In some embodiments, the compound comprising a free nucleophilic moietyis a nucleic acid previously prepared using methods described herein orit is prepared using other known methods of nucleic acid synthesis. Inone embodiment, the compound comprising a free nucleophilic moietycomprises a single nucleoside monomer. In another embodiment, thecompound comprising a free nucleophilic moiety comprises more than onenucleoside unit. In some embodiments, the compound comprising a freenucleophilic moiety is a product of a chain elongation step. In yetother embodiments, the compound comprising a free nucleophilic moiety isan oligomer. In further embodiments, the compound comprising a freenucleophilic moiety is a polymer. In some embodiments the compoundcomprising a free nucleophilic moiety comprises a hydroxyl group as thefree nucleophilic moiety. In some embodiments the compound comprising afree nucleophilic moiety comprises an amino group as the freenucleophilic moiety. In some embodiments the compound comprising a freenucleophilic moiety comprises a thiol group as the free nucleophilicmoiety.

In some embodiments, the compound comprising a free nucleophilic moietycomprises a nucleophilic moiety at any position of the nucleoside sugar.In some embodiments, the nucleophilic moiety is located at the 5′position of the sugar. In some embodiments, the nucleophilic moiety islocated at the 4′ position of the sugar. In other embodiments, thenucleophilic moiety is located at the 3′ position of the sugar. In otherembodiments, the nucleophilic moiety is located at the 2′ position ofthe sugar.

In some embodiments, the compound of Formula 4-1 is a nucleosidecomprising a 5′-OH moiety and is a compound of Formula 4:

In Formula 4, each instance of R² is independently hydrogen,—NR^(d)R^(d), N₃, halogen, alkyl, alkenyl, alkynyl, alkyl-Y alkenyl-Y¹—,alkynyl-Y¹—, aryl-Y¹—, heteroaryl-Y¹—, —OR^(b), or —SR^(c), whereinR^(b) is a blocking moiety.

Y¹ is O, NR^(d), S, or Se.

R^(c) is a blocking group.

Each instance of R^(d) is independently hydrogen, alkyl, alkenyl,alkynyl, aryl, acyl, substituted silyl, carbamate, —P(O)(R^(e))₂, or—HP(O)(R^(e)).

Each instance of R^(e) is independently alkyl, aryl, alkenyl, alkynyl,alkyl-Y²—, alkenyl-Y²—, alkynyl-Y²—, aryl-Y²—, or heteroaryl-Y²—.

Y² is O, NR^(d), or S.

Each instance of Ba is independently a blocked or unblocked adenine,cytosine, guanine, thymine, uracil or modified nucleobase.

m is an integer of 0 to n−1.

n is an integer of 1 to about 200.

O_(A) is connected to a trityl moiety, a silyl moiety, an acetyl moiety,an acyl moiety, an aryl acyl moiety, a linking moiety connected to asolid support or a linking moiety connected to a nucleic acid.

J is O and D is H, or J is S, Se, or BH₃ and D is a chiral ligand C, ora moiety of Formula A:

wherein W₁ and W₂ are independently NHG⁵, OH, or SH.

A is hydrogen, acyl, aryl, alkyl, aralkyl, or silyl moiety.

G¹, G², G³, G⁴, and G⁵ are independently hydrogen, alkyl, aralkyl,cycloalkyl, cycloalkylalkyl, heteroaryl, or aryl, or two of G¹, G², G³,G⁴, and G⁵ are G⁶ which taken together form a saturated, partiallyunsaturated or unsaturated carbocyclic or heteroatom-containing ring ofup to about 20 ring atoms which is monocyclic or polycyclic, fused orunfused and wherein no more than four of G¹, G², G³, G⁴, and G⁵ are G⁶.

In other embodiments, where J is S, Se, or BH₃ and D is a moiety ofFormula A-I:

wherein U₁, U₃, U₂, r, G¹, G², G³, G⁴, W₁, W₂, A are as defined hereinfor Formula 3-I. In an embodiment of Formula A-I, A is hydrogen.

In some embodiments, the nucleoside comprising a 5′-OH moiety of Formula4 is an intermediate from a previous chain elongation cycle as describedherein. In yet other embodiments, the compound of Formula 4 is anintermediate from another known nucleic acid synthetic method. In someembodiments, the compound of Formula 4 is attached to solid support. Inother embodiments, the compound of Formula 4 is not attached to solidsupport and is free in the solvent or solution.

In an embodiment, m is 0 and the compound of formula 4 is a singlenucleoside unit and is considered the first nucleoside of the nucleicacid. In some embodiment, m is 0 and the compound of formula 4 is anucleoside unit attached to another nucleic acid through the 3′-oxygen.In other embodiments, m is greater than 0 and the compound of formula 4is a polymeric or oligomeric nucleic acid comprising a 5′-OH moiety. Inother embodiments, m is greater than 0 and the compound of formula 4 isthe end product of a previous chain elongation cycle. In someembodiments, where m is greater than 0, the compound of formula 4 is anucleoside which is further attached to a nucleotide, through a linkageeither at the 3′ position or at another position on the nucleoside.

Where the compound of formula 4 is attached to another nucleotide ornucleic acid, the phosphate internucleoside backbone linkage include,and are not limited to, 2′ to 5′ phosphorous atom bridges, 3′ to 5′phosphorous atom bridges, 5′ to 3′ phosphorous atom bridges, and the 3′to 2′ phosphorous atom bridges and 4′ to 2′ bridges. The phosphateinternucleoside backbone linkage includes other types of phosphorousatom bridges are also contemplated including, but not limited to,methylene bisphosphonate bridges.

The nucleic acid of Formula 1 comprises the same or differentnucleobases. In some embodiments, the nucleic acid of Formula 1comprises all the same nucleobases. In other embodiments, the nucleicacid of Formula 1 comprises different nucleobases. In other embodiments,the nucleic acid of Formula 1 comprises the naturally occurringnucleobases. In some embodiments, the nucleic acid of Formula 1comprises modified nucleobases. In yet other embodiments, the nucleicacid contain nucleobases that mimic the nucleobase sequence of a nucleicacid found in nature. In some embodiments, the nucleic acid of Formula 1comprises a mixture of naturally occurring nucleobases and modifiednucleobases.

The compound comprising a free nucleophilic moiety is free in solution.In some embodiments, the compound comprising a free nucleophilic moietyis not attached to a solid support. This allows the nucleic acids to besynthesized in solution (liquid phase synthesis or solution phasesynthesis). Alternatively, the compound comprising a free nucleophilicmoiety is pre-attached to another moiety such as a solid support. Insome embodiments, the compound comprising a free nucleophilic moiety isa nucleoside attached to a solid support at the 3′ hydroxyl of thenucleoside. Attachment of the nucleic acid to a solid support allowssynthesis using solid-phase synthesis. During nucleic acid synthesis,the compound attached to a solid support is treated with variousreagents in one or repeated chain elongation cycles to achieve thestepwise elongation of a growing nucleic acid chain with individualnucleic acid units. Purification steps are typically not carried outuntil the fully-assembled nucleic acid sequence is synthesized. Varioustypes of solid support materials are known and used in the synthesis ofnucleic acids, proteins, and oligosaccharides. In some embodiments, thecompound comprising a free nucleophilic moiety is attached to a solidsupport through a linking moiety. In other embodiments, the compoundcomprising a free nucleophilic moiety is attached to a solid supportwithout a linking moiety.

The compound comprising a free nucleophilic moiety comprises a sugar,substitute sugar, or modified sugar. In some embodiments, the sugar is aribose sugar. In some embodiments, the sugar is a deoxyribose sugar. Insome embodiments, compound comprising a free nucleophilic moietycomprises a mixture of a ribose sugar and a deoxyribose sugar. In otherembodiments, the sugar is pentofuranose, pentopyranose, hexopyranosemoieties or mixtures thereof. In further embodiments, the sugarcomprises a closed ring structure, an open structure, or mixturesthereof.

The nucleoside reactant comprising an unprotected-OH moiety may containthe unprotected-OH group at any position on the sugar core. In oneembodiment, an achiral H-phosphonate moiety is condensed with anucleoside comprising a 5′-OH moiety to form the condensed intermediate.In another embodiment, an achiral H-phosphonate moiety is condensed witha nucleoside comprising a 4′-OH moiety to form the condensedintermediate. In another embodiment, an achiral H-phosphonate moiety iscondensed with a nucleoside comprising a 3′-OH moiety to form thecondensed intermediate. In yet another embodiment, an achiralH-phosphonate moiety is condensed with a nucleoside comprising a 2′-OHmoiety to form the condensed intermediate.

In some embodiments, acidifying the condensed intermediate produces acompound of Formula 4 wherein m is at least one. In other embodiments,the condensed intermediate comprises a moiety of Formula A′, which isequivalent to a moiety of Formula A wherein A is hydrogen and wherein G¹and G² are independently alkyl, aralkyl, cycloalkyl, cycloalkylalkyl,heteroaryl, or aryl and G³, G⁴, and G⁵ are independently hydrogen,alkyl, aralkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heteroaryl,or aryl, or two of G¹, G², G³, G⁴, and G⁵ are G⁶ which taken togetherform a saturated, partially unsaturated or unsaturated carbocyclic orheteroatom-containing ring of up to about 20 ring atoms which ismonocyclic or polycyclic, fused or unfused and wherein no more than fourof G¹, G², G³, G⁴, and G⁵ are G⁶.

Detailed Discussion of the Methods of Synthesis.

The extension of the nucleic acid chain can be performed in the 3′ to 5′direction. In one embodiment, the nucleic acid is synthesized from thefree hydroxyl at the 5′-end in repetitive cycles of chemical reactions.Alternatively, the extension of the nucleic acid chain can be performedin the 5′ to 3′ direction. In an embodiment, the nucleic acid issynthesized from the free hydroxyl at the 3′-end in repetitive cycles ofchemical reactions.

One embodiment of the method of synthesis of the nucleic acid is shownin Scheme 5 (Route A). It is understood that the methods herein are notlimited to the scheme, its sequence of events, or its intermediates asillustrated. In one embodiment, described in Scheme 5, an achiralH-phosphonate of Formula 2 is treated with a condensing reagent to forman intermediate of structure II. In one embodiment, an activatingreagent is added to the reaction mixture during the condensation step.Use of an activating reagent is dependent on reaction conditions such assolvents that are used for the reaction. The intermediate of structureII is not isolated and is treated in the same pot with a chiral reagentto form a chiral intermediate of structure III. The intermediate ofstructure III is not isolated and undergoes a reaction in the same potwith a nucleoside or modified nucleoside of structure IV to provide achiral phosphite compound of structure V. In some embodiments, structureV is extracted into a solvent to separate it from side products,impurities, and/or reagents. In other embodiments, when the method isperformed via solid phase synthesis, the solid support comprising thecompound of structure V is filtered away from side products, impurities,and/or reagents. If the final nucleic acid is larger than a dimer, thechiral auxiliary in the compound of structure V is capped with ablocking group to provide a compound of structure VI. If the finalnucleic acid is a dimer, then the capping step is not necessary. Thecompound of structure VI is modified by reaction with an electrophile toprovide a compound of structure VII. The modified and capped condensedintermediate of structure VII is deblocked to remove the blocking groupat the 5′-end of the growing nucleic acid chain to provide a compound ofstructure IV. The compound of structure IV is optionally allowed tore-enter the chain elongation cycle to form a condensed intermediate, acapped condensed intermediate, a modified capped condensed intermediate,and a 5′-deprotected modified capped intermediate. Following at leastone round of chain elongation cycle, the 5′-deprotected modified cappedintermediate is further deblocked by removal of the chiral auxiliaryligand and other protecting groups, e.g., nucleobase, modifiednucleobase, sugar and modified sugar protecting groups, to provide anucleic acid of Formula 1. In other embodiments, the nucleosidecomprising a 5′-OH moiety is an intermediate from a previous chainelongation cycle as described herein. In yet other embodiments, thenucleoside comprising a 5′-OH moiety is an intermediate obtained fromanother known nucleic acid synthetic method. After a cycle of synthesiswith the first nucleoside, nucleosides, nucleotides, or nucleic acidsthat contain an unprotected —OH moiety can be used for subsequentelongation cycles. In embodiments where a solid support is used, thephosphorus-atom modified nucleic acid is then cleaved from the solidsupport. In certain embodiments, the nucleic acids is left attached onthe solid support for purification purposes and then cleaved from thesolid support following purification. In one embodiment, the synthesisdescribed in Scheme 5 (Route A) is useful when the G¹ and G² positionsof the chiral auxiliary ligand of Formula A are hydrogen. In yet otherembodiments, the compounds of structure III-VII comprise a moiety ofFormula A-I instead of a moiety of Formula A.

In another embodiment, described in Scheme 6 (Route B), an achiralH-phosphonate of Formula 2 is treated with a condensing reagent to forman intermediate of structure II. In one embodiment, an activatingreagent is added to the reaction mixture during the condensation step.Use of an activating reagent is dependent on reaction conditions such assolvents that are used for the reaction. The intermediate of structureII is not isolated and is treated in the same pot with a chiral reagentto form a chiral intermediate of structure III. The intermediate ofstructure III is not isolated and undergoes a reaction in the same potwith a nucleoside or modified nucleoside of structure IX to provide achiral phosphite compound of structure X. In some embodiments, structureX is extracted into a solvent to separate it from side products,impurities, and/or reagents. In other embodiments, when the method isperformed via solid phase synthesis, the solid support comprising thecompound of structure X is filtered away from side products, impurities,and/or reagents. The compound of structure X is treated with an acid toremove the blocking group at the 5′-end of the growing nucleic acidchain (structure XI). The acidification step also removes the chiralauxiliary ligand to provide a compound of structure IX. The 5′-deblockedintermediate is optionally allowed to re-enter the chain elongationcycle to form a condensed intermediate containing a blocked 5′-end,which is then acidified to remove the 5′-end blocking group and chiralauxiliary ligand. Following at least one round of chain elongationcycle, the 5′-deprotected intermediate undergoes a modifying step tointroduce a moiety X bonded to each of the phosphorus atoms to provide acompound of structure XII. The modified intermediate is deblocked byremoval of remaining protecting groups, e.g., nucleobase, modifiednucleobase, sugar or modified sugar protecting groups are removed, toprovide a nucleic acid of Formula 1. In other embodiments, thenucleoside comprising a 5′-OH moiety is an intermediate from a previouschain elongation cycle as described herein. In yet other embodiments,the nucleoside comprising a 5′-OH moiety is an intermediate obtainedfrom another known nucleic acid synthetic method. After a cycle ofsynthesis with the first nucleoside, the nucleoside, nucleotide, ornucleic acid that contain an unprotected —OH moiety can be used forsubsequent elongation cycles. In embodiments where a solid support isused, the phosphorus-atom modified nucleic acid is then cleaved from thesolid support. In certain embodiments, the nucleic acids is leftattached on the solid support for purification purposes and then cleavedfrom the solid support following purification. In one embodiment, thesynthesis described in Scheme 6 (Route B) is useful when the G¹ and G²positions of the chiral auxiliary ligand of Formula A are not hydrogen.In some embodiments, the compounds of structures III, X, and XI comprisea moiety of Formula A-I in place of a moiety of Formula A.

Reverse 5′ to 3′ Nucleic Acid Synthesis.

A nucleic acid comprising a chiral X-phosphonate moiety of Formula 1alternatively is synthesized from the 5′ to 3′ direction. In embodimentswhere a solid support is used, the nucleic acid is attached to the solidsupport through its 5′ end of the growing nucleic acid, therebypresenting its 3′ group for reaction, including enzymatic reaction (e.g.ligation and polymerization). In some embodiments, this orientation isengineered by preparing nucleoside monomers comprising an achiralH-phosphonate moiety at the 5′ position and protected hydroxyl group atthe 3′ position. In an embodiment, the nucleic acid is synthesizedaccording to Scheme 7. In Scheme 7,—R⁴ is —OR^(b) as defined above or,in the last cycle of synthesis, is R⁴, which is equivalent to R¹ asdefined herein.

In the embodiment described in Scheme 7, an achiral H-phosphonate ofstructure I_(r) is treated with a condensing reagent to form anintermediate of structure II_(r). The intermediate of structure II_(r)is not isolated and is treated in the same pot with a chiral reagent toform an intermediate of structure III_(r). In one embodiment, anactivating reagent is used during the condensation step. Use of anactivating reagent is dependent on reaction conditions such as solventsthat are used for the reaction. The intermediate of structure III_(r) isnot isolated and undergoes a reaction in the same pot with a nucleosideor modified nucleoside of structure XIII to provide a chiral phosphitecompound of structure XIV. In some embodiments, structure XIV isextracted into a solvent to separate it from side products, impurities,and/or reagents. In other embodiments, when the method is performed viasolid phase synthesis, the solid support comprising the compound ofstructure XIV is filtered away from side products, impurities, and/orreagents. The compound of structure XIV is treated with an acid toremove the blocking group at the 3′-end of the growing nucleic acidchain (structure XV). The acidification step also removes the chiralauxiliary ligand to provide a compound of structure XIII. The3′-deblocked intermediate is optionally allowed to re-enter the chainelongation cycle to form a condensed intermediate containing a blocked3′-end, which is then acidified to remove the 3′-end blocking group andchiral auxillary ligand. Following at least one round of chainelongation cycle, the 3′-deprotected intermediate undergoes a modifyingstep to introduce a moiety X bonded to each of the phosphorus atoms toprovide a compound of structure XVI. The modified intermediate isdeblocked by removal of remaining protecting groups, e.g., nucleobase,modified nucleobase, sugar or modified sugar protecting groups areremoved, to provide a nucleic acid of Formula 1. In other embodiments,the nucleoside comprising a 3′-OH moiety is an intermediate from aprevious chain elongation cycle as described herein. In yet otherembodiments, the nucleoside comprising a 3′-OH moiety is an intermediateobtained from another known nucleic acid synthetic method. After a cycleof synthesis with the first nucleoside, nucleosides, nucleotides, ornucleic acids that contain an unprotected —OH moiety can be used forsubsequent elongation cycles. In embodiments where a solid support isused, the phosphorus-atom modified nucleic acid can then be cleaved fromthe solid support, located at the 5′ end. In certain embodiments, thenucleic acids can optionally be left attached on the solid support forpurification purposes and then cleaved from the solid support followingpurification. In one aspect, the synthesis described in Scheme 7 isuseful when both of the G¹ and G² position of the chiral auxiliaryligand of Formula A are not hydrogen. The reverse 5′ to 3′ synthesis canbe accomplished using the same starting materials in Scheme 7 in amechanism analogous to steps in Route A. In some embodiments, thecompounds of structures III_(r), XIV, and XV comprise a moiety ofFormula A-I in place of a moiety of Formula A.

Chain Elongation Cycle.

The stereoselective synthesis of a phosphorus atom modified nucleic acidcomprises a chain elongation cycle. The chain elongation cycle beginswith a condensation reaction between a compound that is the next unit(e.g. molecular comprising an achiral H-phosphonate moiety) to be addedto the nucleic acid and another compound comprising a free nucleophilicmoiety (e.g. hydroxyl moiety). In some embodiments, the compoundcomprising a free nucleophilic moiety is a monomer nucleoside. In otherembodiments, compound comprising a free nucleophilic moiety is a nucleicacid oligomer or polymer from a previous chain elongation cycle asdescribed herein. In other embodiments, compound comprising a freenucleophilic moiety is a nucleic acid oligomer or polymer from a chainelongation cycle performed using other methods known in the art.

The number of rounds of chain elongation cycles is determined by thelength of the nucleic acid being synthesized. In some embodiments thechain elongation cycle occurs once. In other embodiments, the chainelongation cycle is repeated more than once to achieve the stepwiseelongation of a growing oligonucleotide chain with individual nucleotideunits.

In one embodiment, one round of chain elongation cycle is needed if anucleic acid is a dimer. In another embodiment, 9 rounds of the chainelongation cycle are needed if a nucleic acid comprises ten nucleosideunits. In yet another embodiment, 20 rounds of the chain elongationcycle are needed if 20 additional nucleoside units are to be added to apre-synthesized nucleic acid chain. It will be evident to those skilledin art that the number of chain elongation cycles can be adjusted forthe target length of the nucleic acid. The nucleic acids synthesized bythe methods herein are not limited by the number of chain elongationcycles as described herein.

Modification of the Condensed Intermediate Obtained Via Route A toIntroduce an X-Phosphonate Moiety.

In the compound of Formula 5, R¹ is —NR^(d)R^(d), —N₃, halogen,hydrogen, alkyl, alkenyl, alkynyl, alkyl-Y¹—, alkenyl-Y¹—, alkynyl-Y¹—,aryl-Y¹—, heteroaryl-Y¹—, —P(O)(R^(e))₂, or —HP(O)(R^(e)), —OR^(a), or—SR^(c).

Y¹ is O, NR^(d), S, or Se; R^(a) is a blocking moiety.

R^(c) is a blocking group.

Each instance of R^(d) is independently hydrogen, alkyl, alkenyl,alkynyl, aryl, acyl, substituted silyl, carbamate, —P(O)(R^(e))₂, or—HP(O)(R^(e)).

Each instance of R^(e) is independently alkyl, aryl, alkenyl, alkynyl,alkyl-Y²—, alkenyl-Y²—, alkynyl-Y²—, aryl-Y²—, or heteroaryl-Y²—.

Y² is O, NR^(d), or S.

Each instance of R² is independently hydrogen, —NR^(d)R^(d), N₃,halogen, alkyl, alkenyl, alkynyl, alkyl-Y¹—, alkenyl-Y¹—, alkynyl-Y¹—,aryl-Y¹—, heteroaryl-Y¹—, —OR^(b), or —SR^(c), wherein R^(b) is ablocking moiety.

Each instance of Ba is independently a blocked or unblocked adenine,cytosine, guanine, thymine, uracil, or modified nucleobase.

Each instance of J is S, Se, or BH₃; v is an integer of 1 to n−1.

O_(A) is connected to a linking moiety connected to a solid support or alinking moiety connected to a nucleic acid.

A is an acyl, aryl, alkyl, aralkyl, or silyl moiety; and G¹, G², G³, G⁴,and G⁵ are independently hydrogen, alkyl, aralkyl, cycloalkyl,cycloalkylalkyl, heterocyclyl, heteroaryl, or aryl, or two of G¹, G₂,G₃, G₄, and G⁵ are G⁶ which taken together form a saturated, partiallyunsaturated or unsaturated carbocyclic or heteroatom-containing ring ofup to about 20 ring atoms which is monocyclic or polycyclic, fused orunfused and wherein no more than four of G¹, G², G³, G⁴, and G⁵ are G⁶.

In some embodiments, the compound of Formula 5 comprises a moiety ofFormula A-I attached at the phosphorus atom. In other embodiments, thecompound of Formula 5 comprises a moiety of Formula A attached at thephosphorus atom. In the method illustrated in Route A, the condensedintermediate resulting from addition of a new nucleoside is capped toproduce the compound of structure V and then is modified at thephosphorus to introduce J, which is S, Se, or BH₃, producing a compoundof Formula 5, where v is an integer of 1 to n−1. The compound of Formula5 is either treated to cleave the capped chiral auxiliary and deblockremaining blocking groups or it is subjected to further cycles of chainelongation and phosphorus modification. In the case that the finalnucleic acid is a dimer, capping is not necessary. In one embodiment ofstructure V, A is hydrogen, acyl, aryl, alkyl, aralkyl, or silyl moiety.In one embodiment of Scheme 9, the condensed intermediate resulting fromaddition of a new nucleoside is not capped to produce a compound ofstructure V, where v is 0. This structure V, where v is 0, is thenmodified at the phosphorus to introduce J, which is S, Se, or BH₃,producing a compound of Formula 5, where v is an integer of 0.

In some embodiments, the modifying agent is a sulfur electrophile,selenium electrophile, or boronating agent.

In some embodiments, the sulfur electrophile is a compound having one ofthe following formulas:

S₈ (Formula B), Z²⁴—S—S—Z²⁵, or Z²⁴—S—X—Z²⁵,

wherein Z²⁴ and Z²⁵ are independently alkyl, aminoalkyl, cycloalkyl,heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl,alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide, or thiocarbonyl,or Z²⁴ and Z²⁵ are taken together to form a 3 to 8 membered alicyclic orheterocyclic ring, which may be substituted or unsubstituted; X is SO₂,O, or NR^(f); and R^(f) is hydrogen, alkyl, alkenyl, alkynyl, or aryl.In other embodiments, the sulfur electrophile is a compound of FormulaB, C, D, E, or F:

In other embodiments, the sulfur electrophile is Formula F, Formula E orFormula B.

In some embodiments, the selenium electrophile is a compound having oneof the following formulas:

Se (Formula G), Z²⁶—Se—Se—Z²⁷, or Z²⁶—Se—X—Z²⁷,

wherein Z²⁶ and Z²⁷ are independently alkyl, aminoalkyl, cycloalkyl,heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl,alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide, or thiocarbonyl,or Z²⁶ and Z²⁷ are taken together to form a 3 to 8 membered alicyclic orheterocyclic ring, which may be substituted or unsubstituted; X is SO₂,S, O, or NR^(f); and R^(f) is hydrogen, alkyl, alkenyl, alkynyl, oraryl.

In other embodiments, the selenium electrophile is a compound of FormulaG, H, I, J, K, or L.

In some embodiments, the selenium electrophile is Formula G or FormulaL.

In some embodiments, the boronating agent isborane-N,N-diisopropylethylamine (BH₃.DIPEA), borane-pyridine (BH₃.Py),borane-2-chloropyridine (BH₃.CPy), borane-aniline (BH₃.An),borane-tetrahydrofuran (BH₃.THF), or borane-dimethylsulfide (BH₃.Me₂S),aniline-cyanoborane, triphenyl phosphine-carboalkoxyboranes.

In other embodiments, the boronating agent isborane-N,N-diisopropylethylamine (BH₃.DIPEA), borane-2-chloropyridine(BH₃.CPy), borane-tetrahydrofuran (BH₃.THF), or borane-dimethylsulfide(BH₃.Me₂S).

In further embodiments, after modification of the condensed intermediateobtained via Route A, the compound of Formula 5 is deblocked at the R¹position to produce a compound of Formula 4, wherein m is at least 1, Jis S, Se, or BH₃ and D is a moiety of Formula A. In some embodiments,following deblocking of R¹, a compound of Formula 4 is produced whereinD is a moiety of Formula A-I. The compound of Formula 4 is reacted witha nucleoside of structure III to produce a condensed intermediate. Thestep of converting the condensed intermediate comprises capping thecondensed intermediate and modifying the capped condensed intermediateto produce a compound of Formula 5. In some embodiments of the compoundof Formula 5, v is greater than 2 and less than about 200. Deblocking atthe R¹ position, reacting with a nucleoside of structure III, capping,and modifying is optionally repeated to form a compound of Formula 5wherein v is increased by 1 integer. In some embodiments of the compoundof Formula 5, v is greater than 3 and less than about 200.

In further embodiments, the compound of Formula 5 is converted to thecompound of Formula 1 where in some embodiments, each Ba moiety isunblocked. In other embodiments, the compound of Formula 5 is convertedto the compound of Formula 1 wherein not all Ba moieties are unblocked.

R¹ is —OH, —SH, —NR^(d)R^(d), —N₃, halogen, hydrogen, alkyl, alkenyl,alkynyl, alkyl-Y¹—, alkenyl-Y¹—, alkynyl-Y¹—, aryl-Y¹—, heteroaryl-Y¹—,—P(O)(R^(e))₂, —HP(O)(R^(e)), —OR^(a), or —SR^(c); where Y¹ is O,NR^(d), S, or Se, R^(a) is a blocking moiety, and R^(c) is a blockinggroup. In some embodiments, R¹ is deblocked. In yet other embodiments,R¹ remains blocked.

Each instance of R^(d) is independently hydrogen, alkyl, alkenyl,alkynyl, aryl, substituted silyl, carbamate, —P(O)(R^(e))₂, or—HP(O)(R^(e)), and each instance of R^(e) is independently hydrogen,alkyl, aryl, alkenyl, alkynyl, alkyl-Y²—, alkenyl-Y²—, alkynyl-Y²—,aryl-Y²—, or heteroaryl-Y²—, or a cation which is Na⁺¹, Li⁺¹, or K⁺¹,where Y² is O, NR^(d), or S.

Each instance of R² is independently hydrogen, —OH, —SH, —NR^(d)R^(d),N₃, halogen, alkyl, alkenyl, alkynyl, alkyl-Y¹—, alkenyl-Y¹—,alkynyl-Y¹—, aryl-Y¹—, heteroaryl-Y¹—.

In some embodiments, R³ is H. In other embodiments, R³ is a blockinggroup or a linking moiety connected to solid support, nucleoside,nucleotide, or nucleic acid. In some embodiments, each instance of X isindependently —S⁻Z⁺, —Se⁻Z⁺, or —BH₃ ⁻Z⁺; and Z⁺ is ammonium ion,alkylammonium ion, heteroaromatic iminium ion, or heterocyclic iminiumion, any of which is primary, secondary, tertiary or quaternary, or Z⁺is a monovalent metal ion.

Modification of the Compound of Formula 4 Obtained Via Route B toIntroduce an X-Phosphonate Moiety.

Methods used to modify the compound of Formula 4 obtained via Route Bare illustrated in Reaction Schemes 9a and 9b. Phosphonate and phosphiteare known to tautomerize and exist in equilibrium. The phosphitetautomer is less stable than the phosphonate tautomer. Equilibrium liestoward the phosphonate tautomer under neutral conditions due to the verystrong P═O bond. Under acidic conditions, the phosphoryl group of thephosphonate becomes reversibly protonated. Cleavage of the P—H bond inthe intermediate occurs slowly to produce the phosphite intermediate.Structure IX is then modified to form structure XII, using reagentsshown in Reaction Schemes 9a and 9b.

In some embodiments, the modifying step is performed by reactingstructure IX with a halogenating reagent followed by reacting with anucleophile (Scheme 9a). In specific embodiments, the halogenatingreagent is CCl₄, CBr₄, CI₄, Cl₂, Br₂, I₂, sulfuryl chloride (SO₂Cl₂),phosgene, bis(trichloromethyl)carbonate (BTC), sulfur monochloride,sulfur dichloride, chloramine, CuCl₂, N-chlorosuccinimide (NCS),N-bromosuccinimide (NBS), or N-iodosuccinimide (NIS). In other specificembodiments, the halogenating reagent is CCl₄, CBr₄, Cl₂, sulfurylchloride (SO₂Cl₂), or N-chlorosuccinimide (NCS). In some embodiments,the nucleophile is primary or secondary amines, alcohols, or thiols. Inother embodiments, the nucleophile is NR^(f)R^(f)H, R^(f)OH, or R^(f)SH,wherein R^(f) is hydrogen, alkyl, alkenyl, alkenyl, or aryl, and atleast one of R^(f) of NR^(f)R^(f)H is not hydrogen.

The modifying step can also be performed by reacting structure IX with asilylating reagent followed by reaction with a sulfur electrophile, aselenium electrophile, a boronating agent, an alkylating agent, analdehyde, or an acylating agent (Scheme 9b).

In specific embodiments, the silylating reagent is chlorotrimethylsilane(TMS-Cl), triisopropylsilylchloride (TIPS-Cl),t-butyldimethylsilylchloride (TBDMS-Cl), t-butyldiphenylsilylchloride(TBDPS-Cl), 1,1,1,3,3,3-hexamethyldisilazane (HMDS),N-trimethylsilyldimethylamine (TMSDMA), N-trimethylsilyldiethylamine(TMSDEA), N-trimethylsilylacetamide (TMSA),N,O-bis(trimethylsilyl)acetamide (BSA), orN,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA).

In other specific embodiments, the sulfur electrophile is a compoundhaving one of the following formulas:

S₈ (Formula B), Z²⁴—S—S—Z²⁵, or Z²⁴—S—X—Z²⁵,

wherein Z²⁴ and Z²⁵ are independently alkyl, aminoalkyl, cycloalkyl,heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl,alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide, or thiocarbonyl,or Z²⁴ and Z²⁵ are taken together to form a 3 to 8 membered alicyclic orheterocyclic ring, which may be substituted or unsubstituted; X is SO₂,O, or NR^(f); and R^(f) is hydrogen, alkyl, alkenyl, alkynyl, or aryl.In other embodiments, the sulfur electrophile is a compound of FormulaB, C, D, E, or F:

In other embodiments, the sulfur electrophile is Formula F, Formula E orFormula B.

In some embodiments, selenium electrophile is a compound having one ofthe following formulas:

Se (Formula G), Z²⁶—Se—Se—Z²⁷, or Z²⁶—Se—X—Z²⁷,

wherein Z²⁶ and Z²⁷ are independently alkyl, aminoalkyl, cycloalkyl,heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl,alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide, or thiocarbonyl,or Z²⁶ and Z²⁷ are taken together to form a 3 to 8 membered alicyclic orheterocyclic ring, which may be substituted or unsubstituted; X is SO₂,S, O, or NR^(f); and R^(f) is hydrogen, alkyl, alkenyl, alkynyl, oraryl.

In other embodiments, the selenium electrophile is a compound of FormulaG, H, I, J, K, or L.

In some embodiments, the selenium electrophile is Formula G or FormulaL.

In some embodiments, the boronating agent isborane-N,N-diisopropylethylamine (BH₃.DIPEA), borane-pyridine (BH₃.Py),borane-2-chloropyridine (BH₃.CPy), borane-aniline (BH₃.An),borane-tetrahydrofuran (BH₃.THF), or borane-dimethylsulfide (BH₃.Me₂S),aniline-cyanoborane, triphenyl phosphine-carboalkoxyboranes. In otherembodiments, the boronating agent is borane-N,N-diisopropylethylamine(BH₃.DIPEA), borane-2-chloropyridine (BH₃.CPy), borane-tetrahydrofuran(BH₃.THF), or borane-dimethylsulfide (BH₃.Me₂S).

In other embodiments, the alkylating agent is an alkyl halide, alkenylhalide, alkynyl halide, alkyl sulfonate, alkenyl sulfonate, or alkynylsulfonate.

In other embodiments, the aldehyde is (para)-formaldehyde, alkylaldehyde, alkenyl aldehyde, alkynyl aldehyde, or aryl aldehyde.

In yet other embodiments, the acylating agent is a compound of Formula Mor N:

wherein G⁷ is alkyl, cycloalkyl, heterocyclic, cycloalkylalkyl,heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy, or heteroaryloxy;and M is F, Cl, Br, I, 3-nitro-1,2,4-triazole, imidazole, alkyltriazole,tetrazole, pentafluorobenzene, or 1-hydroxybenzotriazole.

In further embodiments, after acidifying, the compound of Formula 4wherein m is at least one, J is O, and D is H, is reacted with anucleoside of structure III to form a condensed intermediate, which isconverted by acidifying to produce a compound of Formula 4 wherein m isat least 2 and less than about 200; J is O, and D is H. In otherembodiments, the compound of Formula 4 is optionally further reactedwith a nucleoside of structure III to form a condensed intermediatefollowed by acidification. Reaction with the nucleoside of structure IIIand acidification is repeated until a desired number of units in thegrowing chain is achieved. In some embodiments, a compound of Formula 4is produced wherein m is increased by 1 integer. In some embodiments, acompound of Formula 4 wherein m is greater than 2 and less than about200 is produced. In some embodiments, the condensed intermediatecomprises a moiety of Formula A-I in place of a moiety of Formula A.

In further embodiments, the compound of Formula 4 is modified tointroduce an X moiety thereby producing a compound of Formula 1. In anembodiment of the compound of Formula 1, R³ is a blocking group or alinking moiety connected to a solid support. In other embodiments, R¹ isdeblocked. In yet other embodiments, the compound of Formula 1 istreated such that R¹ remains blocked. In yet further embodiments, thecompound of Formula 1 is treated such that R¹ is —OH, —SH, —NR^(d)R^(d),—N₃, halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Y¹—, alkenyl-Y¹—,alkynyl-Y¹—, heteroaryl-Y¹—, —P(O)(R^(e))₂, —HP(O)(R^(e)), —OR^(a), or—SR^(c); where Y¹ is O, NR^(d), S, or Se, R^(a) is a blocking moiety,and R^(c) is a blocking group, each instance of R^(d) is independentlyhydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl,carbamate, —P(O)(R^(e))₂, or —HP(O)(R^(e)), and each instance of R^(e)is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y²—,alkenyl-Y²—, alkynyl-Y²—, aryl-Y²—, or heteroaryl-Y²—, or a cation whichis Na⁺¹, Li⁺¹, or K⁺¹, where Y² is O, NR^(d), or S.

Each instance of R² is independently hydrogen, —OH, —SH, —NR^(d)R^(d),N₃, halogen, alkyl, alkenyl, alkynyl, alkyl-Y¹—, alkenyl-Y¹—,alkynyl-Y¹—, aryl-Y¹—, heteroaryl-Y¹—. In some embodiments, R² isdeblocked. In yet other embodiments, R² remains blocked.

In some embodiments, each Ba moiety is unblocked. In other embodiments,not all Ba moieties are unblocked. In other embodiments, R³ is H. Insome embodiments, R³ is a blocking group or a linking moiety connectedto solid support, nucleoside, nucleotide, or nucleic acid. In someembodiments, each instance of X is independently alkyl, alkoxy, aryl,alkylthio, acyl, —NR^(f)R^(f), alkenyloxy, alkynyloxy, alkenylthio,alkynylthio, —S⁻Z⁺, or —BH₃ ⁻Z⁺; each instance of R^(f) is independentlyhydrogen, alkyl, alkenyl, alkynyl, or aryl; Z⁺ is ammonium ion,alkylammonium ion, heteroaromatic iminium ion, or heterocyclic iminiumion, any of which is primary, secondary, tertiary or quaternary, or Z⁺is a monovalent metal ion.

Reaction Conditions and Reagents Used in the Methods of the InventionConditions

The steps of reacting a molecule comprising an achiral H-phosphonatemoiety and a nucleoside comprising a 5′-OH moiety to form a condensedintermediate can occur without isolating any intermediates. In someembodiments, the steps of reacting a molecule comprising an achiralH-phosphonate moiety and a nucleoside comprising a 5′-OH moiety to forma condensed intermediate occurs is a one-pot reaction. In an embodiment,a molecule comprising an achiral H-phosphonate moiety, condensingreagent, chiral reagent, and compound comprising a free nucleophilicmoiety are added to the reaction mixture at different times. In anotherembodiment, a molecule comprising an achiral H-phosphonate moiety,condensing reagent, and chiral reagent are present in the same reactionvessel or same pot. In another embodiment, a molecule comprising anachiral H-phosphonate moiety, condensing reagent, chiral reagent, andcompound comprising a free nucleophilic moiety are present in the samereaction or same pot. This allows the reaction to be performed withoutisolation of intermediates and eliminates time-consuming steps,resulting in an economical and efficient synthesis. In specificembodiments, the achiral H-phosphonate, condensing reagent, chiral aminoalcohol, 5′-OH nucleoside are present at the same time in a reaction. Ina further embodiment, the formation of the chiral intermediate forcondensation is formed in situ and is not isolated prior to thecondensation reaction. In another embodiment, a molecule comprising anachiral H-phosphonate moiety has been activated by reaction with acondensing reagent, chiral reagent in a different reaction vessel fromthat used when reacting the chiral intermediate with the compoundcomprising a free 5′-OH moiety. In an embodiment, an activating reagentis added during the condensation step. In one embodiment, an activatingreagent is added after achiral H-phosphonate moiety, condensing reagent,and chiral reagent have already been mixed together. In anotherembodiment, an activating reagent is added together with the achiralH-phosphonate moiety, condensing reagent, and chiral reagent. Dependingon the reaction conditions, an activating reagent can be useful duringthe synthesis, for instance, in the condensation step. For example, ifpyridine is used as the base in the preactivation or condensation step,an activating reagent such as CMPT need not be present since pyridineacts as a nucleophilic catalyst (i.e. activator). If another base, suchas N-cyanomethyl pyrrolidine (CMP), that is not as nucleophilic aspyridine is used in the condensation step, then the use of an activatingreagent, such as CMPT, can be added as an activating reagent.

Synthesis on Solid Support

In some embodiments, the synthesis of the nucleic acid is performed insolution. In other embodiments, the synthesis of the nucleic acid isperformed on solid phase. The reactive groups of a solid support may beunprotected or protected. During oligonucleotide synthesis a solidsupport is treated with various reagents in several synthesis cycles toachieve the stepwise elongation of a growing oligonucleotide chain withindividual nucleotide units. The nucleoside unit at the end of the chainwhich is directly linked to the solid support is termed “the firstnucleoside” as used herein. The first nucleoside is bound to the solidsupport via a linker moiety, i.e. a diradical with covalent bonds toboth the polymer of the solid support and the nucleoside. The linkerstays intact during the synthesis cycles performed to assemble theoligonucleotide chain and is cleaved after the chain assembly toliberate the oligonucleotide from the support.

Solid supports for solid-phase nucleic acid synthesis include thesupports described in, e.g., U.S. Pat. Nos. 4,659,774, 5,141,813,4,458,066; Caruthers U.S. Pat. Nos. 4,415,732, 4,458,066, 4,500,707,4,668,777, 4,973,679, and 5,132,418; Andrus et al. U.S. Pat. Nos.5,047,524, 5,262,530; and Koster U.S. Pat. No. 4,725,677 (reissued asU.S. Pat. No. Re34,069). In some embodiments, the solid phase is anorganic polymer support. In other embodiments, the solid phase is aninorganic polymer support. In some embodiments, the organic polymersupport is polystyrene, aminomethyl polystyrene, a polyethyleneglycol-polystyrene graft copolymer, polyacrylamide, polymethacrylate,polyvinylalcohol, highly cross-linked polymer (HCP), or other syntheticpolymers, carbohydrates such as cellulose and starch or other polymericcarbohydrates, or other organic polymers and any copolymers, compositematerials or combination of the above inorganic or organic materials. Inother embodiments, the inorganic polymer support is silica, alumina,controlled polyglass (CPG), which is a silica-gel support, oraminopropyl CPG. Other useful solid supports include fluorous solidsupports (see e.g., WO/2005/070859), long chain alkylamine (LCAA)controlled pore glass (CPG) solid supports (see e.g., S. P. Adams, K. S.Kavka, E. J. Wykes, S. B. Holder and G. R. Galluppi, J. Am. Chem. Soc.,1983, 105, 661-663; G. R. Gough, M. J. Bruden and P. T. Gilham,Tetrahedron Lett., 1981, 22, 4177-4180). Membrane supports and polymericmembranes (see e.g. Innovation and Perspectives in Solid PhaseSynthesis, Peptides, Proteins and Nucleic Acids, ch 21 pp 157-162, 1994,Ed. Roger Epton and U.S. Pat. No. 4,923,901) are also useful for thesynthesis of nucleic acids. Once formed, a membrane can be chemicallyfunctionalized for use in nucleic acid synthesis. In addition to theattachment of a functional group to the membrane, the use of a linker orspacer group attached to the membrane may be used to minimize sterichindrance between the membrane and the synthesized chain.

Other suitable solid supports include those generally known in the artto be suitable for use in solid phase methodologies, including, forexample, glass sold as Primer™ 200 support, controlled pore glass (CPG),oxalyl-controlled pore glass (see, e.g., Alul, et al., Nucleic AcidsResearch, 1991, 19, 1527), TentaGel Support-an aminopolyethyleneglycolderivatized support (see, e.g., Wright, et al., Tetrahedron Lett., 1993,34, 3373), and Poros-a copolymer of polystyrene/divinylbenzene.

Surface activated polymers have been demonstrated for use in synthesisof natural and modified nucleic acids and proteins on several solidsupports mediums. The solid support material can be any polymer suitablyuniform in porosity, has sufficient amine content, and sufficientlyflexible to undergo any attendant manipulations without losingintegrity. Examples of suitable selected materials include nylon,polypropylene, polyester, polytetrafluoroethylene, polystyrene,polycarbonate, and nitrocellulose. Other materials can serve as thesolid support, depending on the design of the investigator. Inconsideration of some designs, for example, a coated metal, inparticular gold or platinum can be selected (see e.g., US publicationNo. 20010055761). In one embodiment of oligonucleotide synthesis, forexample, a nucleoside is anchored to a solid support which isfunctionalized with hydroxyl or amino residues. Alternatively, the solidsupport is derivatized to provide an acid labile trialkoxytrityl group,such as a trimethoxytrityl group (TMT). Without being bound by theory,it is expected that the presence of the trialkoxytrityl protecting groupwill permit initial detritylation under conditions commonly used on DNAsynthesizers. For a faster release of oligonucleotide material insolution with aqueous ammonia, a diglycoate linker is optionallyintroduced onto the support.

Linking Moiety

A linking moiety or linker is optionally used to connect the solidsupport to the compound comprising a free nucleophilic moiety. Suitablelinkers are known such as short molecules which serve to connect a solidsupport to functional groups (e.g., hydroxyl groups) of initialnucleosides molecules in solid phase synthetic techniques. In someembodiments, the linking moiety is a succinamic acid linker, or asuccinate linker (—CO—CH₂—CH₂—CO—), or an oxalyl linker (—CO—CO—). Inother embodiments, the linking moiety and the nucleoside are bondedtogether through an ester bond. In other embodiments, the linking moietyand the nucleoside are bonded together through an amide bond. In furtherembodiments, the linking moiety connects the nucleoside to anothernucleotide or nucleic acid. Suitable linkers are disclosed in, forexample, Oligonucleotides And Analogues A Practical Approach, Ekstein,F. Ed., IRL Press, N.Y., 1991, Chapter 1.

A linker moiety is used to connect the compound comprising a freenucleophilic moiety to another nucleoside, nucleotide, or nucleic acid.In some embodiments, the linking moiety is a phosphodiester linkage. Inother embodiments, the linking moiety is an H-phosphonate moiety. In yetother embodiments, the linking moiety is an X-phosphonate moiety.

Solvents for Synthesis

Synthesis of the nucleic acids is performed in an aprotic organicsolvent. In some embodiments, the solvent is acetonitrile, pyridine,tetrahydrofuran, or dichloromethane. In some embodiments, when theaprotic organic solvent is not basic, a base is present in the reactingstep. In some embodiments where a base is present, the base is pyridine,quinoline, or N,N-dimethylaniline or N-cyanomethylpyrrolidine. Otherexamples of bases include pyrrolidine, piperidine, N-methyl pyrrolidine,pyridine, quinoline, N,N-dimethylaminopyridine (DMAP),N,N-dimethylaniline or N-cyanomethylpyrrolidine. In some embodiments ofthe method, the base is

wherein Z²² and Z²³ are independently alkyl, aminoalkyl, cycloalkyl,heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl,alkyloxy, aryloxy, or heteroaryloxy, or wherein any of Z²² and Z²³ aretaken together to form a 3 to 10 membered alicyclic or heterocyclicring. In some embodiments of the method, the base isN-cyanomethylpyrrolidine. In some embodiments, the aprotic organicsolvent is anhydrous. In other embodiments, the anhydrous aproticorganic solvent is freshly distilled. In some embodiments, the freshlydistilled anhydrous aprotic organic solvent is pyridine. In otherembodiments, the freshly distilled anhydrous aprotic organic solvent istetrahydrofuran. In other embodiments, the freshly distilled anhydrousaprotic organic solvent is acetonitrile. The solvent can be acombination of 2 or more solvents. Depending on which solvent is usedfor the synthesis, addition of an activating reagent is useful.

Acidification Conditions to Remove Blocking Groups.

Acidification to remove blocking groups is accomplished by a Brønstedacid or Lewis acid. In some embodiments, acidification is used to removeR¹ blocking groups. Useful Brønsted acids are carboxylic acids,alkylsulfonic acids, arylsulfonic acids, phosphoric acid and itsderivatives, phosphonic acid and its derivatives, alkyl phosphonic acidsand their derivatives, aryl phosphonic acids and their derivatives,phosphinic acid, dialkyl phosphinic acids, and diaryl phosphinic acidswhich have a pKa (25° C. in water) value of −0.6 (trifluoroacetic acid)to 4.76 (acetic acid) in an organic solvent or water (in the case of 80%acetic acid). The concentration of the acid (1 to 80%) used in theacidification step depends on the acidity of the acid. Consideration tothe acid strength must be taken into account as strong acid conditionswill result in depurination/depyrimidination, wherein purinyl orpyrimidinyl bases are cleaved from ribose ring.

In some embodiments, acidification is accomplished by a Lewis acid in anorganic solvent. Useful Lewis acids are ZnX₂ wherein X is Cl, Br, I, orCF₃SO₃.

In some embodiments, the acidifying comprises adding an amount of aBrønsted or Lewis acid effective to convert the condensed intermediateinto the compound of Formula 4 without removing purine moieties from thecondensed intermediate.

Acids that are useful in the acidifying step also include, but are notlimited to 10% phosphoric acid in an organic solvent, 10% hydrochloricacid in an organic solvent, 1% trifluoroacetic acid in an organicsolvent, 3% dichloroacetic acid in an organic solvent or 80% acetic acidin water. The concentration of any Brønsted or Lewis acid used in theprocess is selected such that the concentration of the acid does notexceed a concentration that causes cleavage of the nucleobase from thesugar moiety.

In some embodiments, acidification comprises adding 1% trifluoroaceticacid in an organic solvent. In some embodiments, acidification comprisesadding about 0.1% to about 8% trifluoroacetic acid in an organicsolvent. In other embodiments, acidification comprises adding 3%dichloroacetic acid in an organic solvent. In other embodiments,acidification comprises adding about 0.1% to about 10% dichloroaceticacid in an organic solvent. In yet other embodiments, acidificationcomprises adding 3% trichloroacetic acid in an organic solvent. In yetother embodiments, acidification comprises adding about 0.1% to about10% trichloroacetic acid in an organic solvent. In some embodiments,acidification comprises adding 80% acetic acid in water. In someembodiments, acidification comprises adding about 50% to about 90%, orabout 50% to about 80%, about 50% to about 70%, about 50% to about 60%,about 70% to about 90% acetic acid in water. In some embodiments, theacidification comprises the further addition of cation scavengers to theacidic solvent. In specific embodiments, the cation scavengers can betriethylsilane or triisopropylsilane. In some embodiments, R¹ isdeblocked prior to the step of acidifying the condensed intermediate. Insome embodiments, R¹ is deblocked by acidification, which comprisesadding 1% trifluoroacetic acid in an organic solvent. In someembodiments, R¹ is deblocked by acidification, which comprises adding 3%dichloroacetic acid in an organic solvent. In some embodiments, R¹ isdeblocked by acidification, which comprises adding 3% trichloroaceticacid in an organic solvent.

Removal of Blocking Moieities or Groups.

Functional groups such as hydroxyl or amino moieties which are locatedon nucleobases or sugar moieties are routinely blocked with blocking(protecting) groups (moieties) during synthesis and subsequentlydeblocked. In general, a blocking group renders a chemical functionalityof a molecule inert to specific reaction conditions and can later beremoved from such functionality in a molecule without substantiallydamaging the remainder of the molecule (see e.g., Green and Wuts,Protective Groups in Organic Synthesis, 2^(nd) Ed., John Wiley & Sons,New York, 1991). For example, amino groups can be blocked with nitrogenblocking groups such as phthalimido, 9-fluorenylmethoxycarbonyl (FMOC),triphenylmethylsulfenyl, t-BOC, 4,4′-dimethoxytrityl (DMTr),4-methoxytrityl (MMTr), 9-phenylxanthin-9-yl (Pixyl), trityl (Tr), or9-(p-methoxyphenyl)xanthin-9-yl (MOX). Carboxyl groups can be protectedas acetyl groups. Hydroxy groups can be protected such astetrahydropyranyl (THP), t-butyldimethylsilyl (TBDMS),1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (Ctmp),1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp),1-(2-chloroethoxy)ethyl, 3-methoxy-1,5-dicarbomethoxypentan-3-yl (MDP),bis(2-acetoxyethoxy)methyl (ACE), triisopropylsilyloxymethyl (TOM),1-(2-cyanoethoxy)ethyl (CEE), 2-cyanoethoxymethyl (CEM),[4-(N-dichloroacetyl-N-methylamino)benzyloxy]methyl, 2-cyanoethyl (CN),pivaloyloxymethyl (PivOM), levunyloxymethyl (ALE). Other representativehydroxyl blocking groups have been described (see e.g., Beaucage et al.,Tetrahedron, 1992, 46, 2223). In some embodiments, hydroxyl blockinggroups are acid-labile groups, such as the trityl, monomethoxytrityl,dimethoxytrityl, trimethoxytrityl, 9-phenylxanthin-9-yl (Pixyl) and9-(p-methoxyphenyl)xanthin-9-yl (MOX). Chemical functional groups canalso be blocked by including them in a precursor form. Thus an azidogroup can be considered a blocked form of an amine as the azido group iseasily converted to the amine Further representative protecting groupsutilized in nucleic acid synthesis are known (see e.g. Agrawal et al.,Protocols for Oligonucleotide Conjugates, Eds., Humana Press, NewJersey, 1994, Vol. 26, pp. 1-72).

Various methods are known and used for removal of blocking groups fromthe nucleic acids. In some embodiments, all blocking groups are removed.In other embodiments, the blocking groups are partially removed. In yetother embodiments, reaction conditions can be adjusted to removeblocking groups on certain moieties. In certain embodiments where R² isa blocking group, removal of the blocking group at R² is orthogonal tothe removal of the blocking group at R¹. The blocking groups at R¹ andR² remain intact during the synthesis steps and are collectively removedafter the chain assembly. In some embodiments, the R² blocking group areremoved simultaneously with the cleavage of the nucleic acids from thesolid support and with the removal of the nucleobase blocking groups. Inspecific embodiments, the blocking group at R¹ is removed while theblocking groups at R² and nucleobases remain intact. Blocking groups atR¹ are cleavable on solid supports with an organic base such as aprimary amine, a secondary amine, or a mixture thereof. Deblocking ofthe R¹ position is commonly referred to as front end deprotection.

In an embodiment, the nucleobase blocking groups, if present, arecleavable after the assembly of the respective nucleic acid with anacidic reagent. In another embodiment, one or more of the nucleobaseblocking groups is cleavable under neither acidic nor basic conditions,e.g. cleavable with fluoride salts or hydrofluoric acid complexes. Inyet another embodiment, one or more of the nucleobase blocking groupsare cleavable after the assembly of the respective nucleic acid in thepresence of base or a basic solvent, and wherein the nucleobase blockinggroup is stable to the conditions of the front end deprotection stepwith amines.

In some embodiments, blocking groups for nucleobases are not required.In other embodiments, blocking groups for nucleobases are required. Inyet other embodiments, certain nucleobases require blocking group whileother nucleobases do not require blocking groups. In embodiments wherethe nucleobases are blocked, the blocking groups are either completelyor partially removed under conditions appropriate to remove the blockinggroup at the front end. For example, R¹ can denote OR^(a), wherein R^(a)is acyl, and Ba denotes guanine blocked with an acyl group including,but not limited to isobutyryl, acetyl or 4-(tert-butylphenoxy)acetyl.The acyl groups at R¹ and Ba will be removed or partially removed duringthe same deblocking step.

Reagents Condensing Reagent.

The condensing reagent (C_(R)) useful in the methods of the inventionhas one of the following general formulae: Ar₃PL₂, and (ArO)₃PL_(2,)

wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, Z⁷, Z⁸, Z⁹ and Z¹⁰ are independentlyselected from alkyl, aminoalkyl, cycloalkyl, heterocyclic,cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy,or heteroaryloxy, or wherein any of Z² and Z³, Z⁵ and Z⁶, Z⁷ and Z⁸, Z⁸and Z⁹, Z⁹ and Z⁷, or Z⁷ and Z⁸ and Z⁹ are taken together to form a 3 to20 membered alicyclic or heterocyclic ring; Q⁻ is a counter anion; w isan integer of 0 to 3; L is a leaving group; and Ar is aryl, heteroaryl,and/or one of Ar group is attached to the polymer support.

In some embodiments, the counter ion of the condensing reagent C_(R) isCl⁻, Br⁻, BF₄ ⁻, PF₆ ⁻, TfO⁻, Tf₂N⁻, AsF₆ ⁻, ClO₄ ⁻, or SbF₆ ⁻, whereinTf is CF₃SO₂. In some embodiments, the leaving group of the condensingreagent C_(R) is F, Cl, Br, I, 3-nitro-1,2,4-triazole, imidazole,alkyltriazole, tetrazole, pentafluorobenzene, or 1-hydroxybenzotriazole.

Examples of condensing agents that can be used in the process include,and are not limited to, pentafluorobenzoyl chloride, carbonyldiimidazole(CDI), 1-mesitylenesulfonyl-3-nitrotriazole (MSNT),1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (EDCI-HCl),benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(PyBOP), N,N′-bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BopCl),2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU), andO-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU),DIPCDI; N,N′-bis(2-oxo-3-oxazolidinyl)phosphinic bromide (BopBr),1,3-dimethyl-2-(3-nitro-1,2,4-triazol-1-yl)-2-pyrrolidin-1-yl-1,3,2-diazaphospholidiniumhexafluorophosphate (MNTP),3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphoniumhexafluorophosphate (PyNTP), bromotripyrrolidinophosphoniumhexafluorophosphate (PyBrOP);O-(benzotriazol-1-yl)-N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU); tetramethylfluoroformamidinium hexafluorophosphate (TFFH);(PhO)₃PCl₂, and bis(trichloromethyl)carbonate (BTC). In certainembodiments, the counter ion of the condensing reagent C_(R) is Cl⁻,Br⁻, BF₄ ⁻, PF₆ ⁻, TfO⁻, Tf₂N⁻, AsF₆ ⁻, ClO₄ ⁻, or SbF₆ ⁻, wherein Tf isCF₃SO₂.

In other embodiments of the invention, the condensing reagent is1-(2,4,6-triisopropylbenzenesulfonyl)-5-(pyridin-2-yl) tetrazolide,pivaloyl chloride, bromotrispyrrolidinophosphonium hexafluorophosphate,N,N′-bis(2-oxo-3-oxazolidinyl) phosphinic chloride (BopCl), (PhO)₃PCl₂,or 2-chloro-5,5-dimethyl-2-oxo-1,3,2-dioxaphosphinane,bis(trichloromethyl)carbonate (BTC), or Ph₃PCl₂. In one embodiment, thecondensing reagent is N,N′-bis(2-oxo-3-oxazolidinyl)phosphinic chloride(BopCl). In one embodiment, the condensing reagent isbis(trichloromethyl)carbonate (BTC). In one embodiment, the condensingreagent is Ph₃PCl₂. Other known condensing reagents have been described(see e.g., WO/2006/066260).

In other embodiments, the condensing reagent is1,3-dimethyl-2-(3-nitro-1,2,4-triazol-1-yl)-2-pyrrolidin-1-yl-1,3,2-diazaphospholidiniumhexafluorophosphate (MNTP),3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphoniumhexafluorophosphate (PyNTP), bis(trichloromethyl)carbonate (BTC),(PhO)₃PCl₂, or Ph₃PCl₂.

Activating Reagent.

The activating reagent useful herein should have strong proton-donatingability to be able to activate the chiral intermediate for reaction witha compound comprising a free nucleophilic moiety. In one embodiment, thechiral intermediate is structure III shown in Scheme 5 or 6 or isstructure III, shown in Scheme 7. The activating reagent acts byprotonating the nitrogen atom of structure III or III, when W1 is anitrogen. Use of an activating reagent is dependent on solvents used forthe synthesis.

The activating reagent (A_(R)) useful in the method of the invention hasone of the following general formulae:

Z¹¹, Z¹², Z¹³, Z¹⁴, Z¹⁵, Z¹⁶, Z¹⁷, Z¹⁸, Z¹⁹, Z²⁰, and Z²¹ areindependently hydrogen, alkyl, aminoalkyl, cycloalkyl, heterocyclic,cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy,or heteroaryloxy, or wherein any of Z¹¹ and Z¹², Z¹¹ and Z¹³, Z¹¹ andZ¹⁴, Z¹² and Z¹³, Z¹² and Z¹⁴, Z¹³ and Z¹⁴, Z¹⁵ and Z¹⁶, Z¹⁵ and Z¹⁷,Z¹⁶ and Z¹⁷, Z¹⁸ and Z¹⁹, or Z²⁰ and Z²¹ are taken together to form a 3to 20 membered alicyclic or heterocyclic ring, or to form 5 or 20membered aromatic ring. Q⁻ is a counter ion. In some embodiments of themethod, the counter ion of the activating reagent A_(R) is Cl⁻, Br⁻, BF₄⁻, PF₆ ⁻, TfO⁻, Tf₂N⁻, AsF₆ ⁻, ClO₄ ⁻, or SbF₆ ⁻, wherein Tf is CF₃SO₂.

In some embodiments of the method, the activating reagent is imidazole,4,5-dicyanoimidazole (DCI), 4,5-dichloroimidazole, 1-phenylimidazoliumtriflate (PhIMT), benzimidazolium triflate (BIT), benztriazole,3-nitro-1,2,4-triazole (NT), tetrazole, 5-ethylthiotetrazole,5-(4-nitrophenyl)tetrazole, N-cyanomethylpyrrolidinium triflate (CMPT),N-cyanomethylpiperidinium triflate, N-cyanomethyldimethylammoniumtriflate.

In some embodiments of the method, the activating reagent is4,5-dicyanoimidazole (DCI), 1-phenylimidazolium triflate (PhIMT),benzimidazolium triflate (BIT), 3-nitro-1,2,4-triazole (NT), tetrazole,or N-cyanomethylpyrrolidinium triflate (CMPT).

In some embodiments of the method, the activating reagent isN-cyanomethylpyrrolidinium triflate (CMPT).

Chiral Reagent.

In the methods of the present invention, chiral reagents are used toconfer stereoselectivity in the production of X-phosphonate linkages.Many different chiral auxiliaries may be used in this process which arecompounds of Formula 3-I where W₁ and W₂ are any of —O—, —S—, or —NG⁵-,which are capable of reacting with the H-phosphonate starting material,a compound of Formula 2 to form the chiral intermediate, as shown instructure III of Schemes 5 and 6.

U₁ and U₃ are carbon atoms which are bonded to U₂ if present, or to eachother if r is 0, via a single, double or triple bond. U₂ is —C—, —CG⁸-,—CG⁸G⁸-, —NG⁸-, —N—, —O—, or —S— where r is an integer of 0 to 5 and nomore than two heteroatoms are adjacent. When any one of U₂ is C, atriple bond must be formed between a second instance of U₂, which is C,or to one of U₁ or U₃. Similarly, when any one of U₂ is CG⁸, a doublebond is formed between a second instance of U₂ which is —CG⁸- or —N—, orto one of U₁ or U₃.

For example, in some embodiments, —U₁—(U₂)_(r)—U₃— is —CG³G⁴-CG¹G²-. Insome embodiments, —U₁—(U₂)_(r)—U₃— is —CG³=CG'-. In some embodiments,—U₁—(U₂)_(r)—U₃— is —C≡C—. In some embodiments, —U₁—(U₂)_(r)—U₃— is—CG³=C G⁸-CG¹G²-. In some embodiments, —U₁—(U₂)_(r)—U₃— is—CG³G⁴-O—CG¹G²-. In some embodiments, —U₁—(U₂)_(r)—U₃— is—CG³G⁴-NG⁸-CG¹G²-. In some embodiments, —U₁—(U₂)_(r)—U₃— is—CG³G⁴-N—CG²-. In some embodiments, —U₁—(U₂)_(r)—U₃— is —CG³G⁴-N═CG⁸-CG¹G²-.

G¹, G₂, G₃, G₄, G⁵, and G⁸ are independently hydrogen, alkyl, aralkyl,cycloalkyl, cycloalkylalkyl, heterocyclyl, hetaryl, or aryl, or two ofG¹, G², G³, G⁴, and G⁵ are G⁶ taken together form a saturated, partiallyunsaturated or unsaturated carbocyclic or heteroatom-containing ring ofup to about 20 ring atoms which is monocyclic or polycyclic, and isfused or unfused. In some embodiments, the ring so formed is substitutedby oxo, thioxo, alkyl, alkenyl, alkynyl, heteroaryl, or aryl moieties.In some embodiments, when the ring formed by taking two G⁶ together issubstituted, it is substituted by a moiety which is bulky enough toconfer stereoselectivity during the reaction.

For example, in some embodiments, the ring formed by taking two of G⁶together is cyclopentyl, pyrrolyl, cyclopropyl, cyclohexenyl,cyclopentenyl, tetrahydropyranyl, or piperazinyl.

In some embodiments of the invention, the chiral reagent is a compoundof Formula 3.

In some embodiments of Formula 3, W₁ and W₂ are independently —NG⁵-,—O—, or —S—; G¹, G², G³, G⁴, and G⁵ are independently hydrogen, alkyl,aralkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, hetaryl, or aryl, ortwo of G¹, G², G³, G⁴, and G⁵ are G⁶ taken together form a saturated,partially unsaturated or unsaturated carbocyclic orheteroatom-containing ring of up to about 20 ring atoms which ismonocyclic or polycyclic, fused or unfused, and no more than four of G¹,G², G³, G⁴, and G⁵ are G⁶. Similarly to the compounds of Formula 3′, anyof G¹, G², G³, G⁴, or G⁵ are substituted by oxo, thioxo, alkyl, alkenyl,alkynyl, heteroaryl, or aryl moieties. In some embodiments, suchsubstitution induces stereoselectivity in X-phosphonate production.

In some embodiments of the invention, the chiral reagent has one of thefollowing Formulae:

In some embodiments, the chiral reagent is an aminoalcohol. In someother embodiments, the chiral reagent is an aminothiol. In yet otherembodiments, the chiral reagent is an aminophenol. In some embodiments,the chiral reagent is (S)- and (R)-2-methylamino-1-phenylethanol, (1R,2S)-ephedrine, or (1R, 2S)-2-methylamino-1,2-diphenylethanol.

In other embodiments of the invention the chiral reagent is a compoundof one of the following formulae:

The choice of chiral reagent, for example, the isomer represented byFormula O or its stereoisomer, Formula P, permits the specific controlof the chirality at phosphorus. Thus either a R_(P) or S_(P)configuration can be selected in each synthesis cycle, permittingcontrol of the overall three dimensional structure of the nucleic acidproduct. In some embodiments of the invention, a nucleic acid producthas all R_(P) stereocenters. In some embodiments of the invention, anucleic acid product has all S_(P) stereocenters. In some embodiments,the selection of R_(P) and S, centers is made to confer a specific threedimensional superstructure to the nucleic acid chain.

Nucleobases and Modified Nucleobases

The nucleobase Ba in Formula 1 is a natural nucleobase or a modifiednucleobase derived from natural nucleobases. Examples include, but arenot limited to, uracil, thymine, adenine, cytosine, and guanine havingtheir respective amino groups protected by acyl protecting groups,2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil,2,6-diaminopurine, azacytosine, pyrimidine analogs such aspseudoisocytosine and pseudouracil and other modified nucleobases suchas 8-substituted purines, xanthine, or hypoxanthine (the latter twobeing the natural degradation products). The modified nucleobasesdisclosed in Chiu and Rana, RNA, 2003, 9, 1034-1048, Limbach et al.Nucleic Acids Research, 1994, 22, 2183-2196 and Revankar and Rao,Comprehensive Natural Products Chemistry, vol. 7, 313, are alsocontemplated as Ba moieties of Formula 1.

Compounds represented by the following general formulae are alsocontemplated as modified nucleobases:

In the formulae above, R⁸ is a linear or branched alkyl, aryl, aralkyl,or aryloxylalkyl group having 1 to 15 carbon atoms, including, by way ofexample only, a methyl, isopropyl, phenyl, benzyl, or phenoxymethylgroup; and each of R⁹ and R¹⁰ represents a linear or branched alkylgroup having 1 to 4 carbon atoms.

Modified nucleobases also include expanded-size nucleobases in which oneor more benzene rings has been added. Nucleic base replacementsdescribed in the Glen Research catalog (www.glenresearch.com); Krueger AT et al, Acc. Chem. Res., 2007, 40, 141-150; Kool, E T, Acc. Chem. Res.,2002, 35, 936-943; Benner S. A., et al., Nat. Rev. Genet., 2005, 6,553-543; Romesberg, F. E., et al., Curr. Opin. Chem. Biol., 2003, 7,723-733; Hirao, I., Curr. Opin. Chem. Biol., 2006, 10, 622-627, arecontemplated as useful for the synthesis of the nucleic acids describedherein. Some examples of these expanded-size nucleobases are shownbelow:

Herein, modified nucleobases also encompass structures that are notconsidered nucleobases but are other moieties such as, but not limitedto, corrin- or porphyrin-derived rings. Porphyrin-derived basereplacements have been described in Morales-Rojas, H and Kool, E T, Org.Lett., 2002, 4, 4377-4380. Shown below is an example of aporphyrin-derived ring which can be used as a base replacement:

Other modified nucleobases also include base replacements such as thoseshown below:

Modified nucleobases which are fluorescent are also contemplated.Non-limiting examples of these base replacements include phenanthrene,pyrene, stillbene, isoxanthine, isozanthopterin, terphenyl,terthiophene, benzoterthiophene, coumarin, lumazine, tethered stillbene,benzo-uracil, and naphtho-uracil, as shown below:

The modified nucleobases can be unsubstituted or contain furthersubstitutions such as heteroatoms, alkyl groups, or linking moietiesconnected to fluorescent moieties, biotin or avidin moieties, or otherprotein or peptides. Modified nucleobases also include certain‘universal bases’ that are not nucleobases in the most classical sense,but function similarly to nucleobases. One representative example ofsuch a universal base is 3-nitropyrrole.

In addition to nucleosides of structure IV or IX, other nucleosides canalso be used in the process disclosed herein and include nucleosidesthat incorporate modified nucleobases, or nucleobases covalently boundto modified sugars. Some examples of nucleosides that incorporatemodified nucleobases include 4-acetylcytidine;5-(carboxyhydroxylmethyl)uridine; 2′-O-methylcytidine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyluridine; dihydrouridine;2′-O-methylpseudouridine; beta,D-galactosylqueosine;2′-O-methylguanosine; N⁶-isopentenyladenosine; 1-methyladenosine;1-methylpseudouridine; 1-methylguanosine; 1-methylinosine;2,2-dimethylguanosine; 2-methyladenosine; 2-methylguanosine;N⁷-methylguanosine; 3-methyl-cytidine; 5-methylcytidine;N⁶-methyladenosine; 7-methylguanosine; 5-methylaminoethyl uridine;5-methoxyaminomethyl-2-thiouridine; beta,D-mannosylqueosine;5-methoxycarbonylmethyluridine; 5-methoxyuridine;2-methylthio-N⁶-isopentenyladenosine;N-((9-beta,D-ribofuranosyl-2-methylthiopurine-6-yl)carbamoyl)threonine;N-((9-beta,D-ribofuranosylpurine-6-yl)-N-methylcarbamoyl)threonine;uridine-5-oxyacetic acid methylester; uridine-5-oxyacetic acid (v);pseudouridine; queosine; 2-thiocytidine; 5-methyl-2-thiouridine;2-thiouridine; 4-thiouridine; 5-methyluridine;2′-O-methyl-5-methyluridine; and 2′-O-methyluridine.

In some embodiments, nucleosides include 6′-modified bicyclic nucleosideanalogs that have either (R) or (S)-chirality at the 6′-position andinclude the analogs described in U.S. Pat. No. 7,399,845. In otherembodiments, nucleosides include 5′-modified bicyclic nucleoside analogsthat have either (R) or (S)-chirality at the 5′-position and include theanalogs described in US Patent Application Publication No. 20070287831.

In some embodiments, the nucleobases or modified nucleobases comprisesbiomolecule binding moieties such as antibodies, antibody fragments,biotin, avidin, streptavidin, receptor ligands, or chelating moieties.In other embodiments, Ba is 5-bromouracil, 5-iodouracil, or2,6-diaminopurine. In yet other embodiments, Ba is modified bysubstitution with a fluorescent or biomolecule binding moiety. In someembodiments, the substituent on Ba is a fluorescent moiety. In otherembodiments, the substituent on Ba is biotin or avidin.

Modified Sugars of the Nucleotide/Nucleoside

The most common naturally occurring nucleotides are ribose sugars linkedto the nucleobases adenosine (A), cytosine (C), guanine (G), and thymine(T) or uracil (U). Also contemplated are modified nucleotides whereinthe phosphate group or the modified phosphorous atom moieties in thenucleotides can be linked to various positions of the sugar or modifiedsugar. As non-limiting examples, the phosphate group or the modifiedphosphorous-atom moiety can be linked to the 2′, 3′, 4′ or 5′ hydroxylmoiety of a sugar or modified sugar. Nucleotides that incorporate themodified nucleobases described above can also be used in the processdisclosed herein. In some embodiments, nucleotides or modifiednucleotides comprising an unprotected —OH moiety are used in the processdisclosed herein.

In addition to the ribose moiety described in Schemes 1-4b, othermodified sugars can also be incorporated in the nucleic acids disclosedherein. In some embodiments, the modified sugars contain one or moresubstituents at the 2′ position including one of the following: F; CF₃,CN, N₃, NO, NO₂, O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S— orN-alkynyl; or O-alkyl-O-alkyl, O-alkyl-N-alkyl or N-alkyl-O-alkylwherein the alkyl, alkenyl and alkynyl may be substituted orunsubstituted C₁-C₁₀ alkyl or C₂-C₁₀ alkenyl and alkynyl. Examples ofsubstituents include, and are not limited to, O(CH₂)_(n)OCH₃, andO(CH₂)_(n)NH₂, wherein n is from 1 to about 10, MOE, DMAOE, DMAEOE. Alsocontemplated herein are modified sugars described in WO 2001/088198; andMartin et al., Helv. Chim. Acta, 1995, 78, 486-504. In some embodiments,modified sugars comprise substituted silyl groups, an RNA cleavinggroup, a reporter group, a fluorescent label, an intercalator, a groupfor improving the pharmacokinetic properties of a nucleic acid, or agroup for improving the pharmacodynamic properties of a nucleic acid,and other substituents having similar properties. The modifications maybe made at the at the 2′, 3′, 4′, 5′, or 6′ positions of the sugar ormodified sugar, including the 3′ position of the sugar on the3′-terminal nucleotide or in the 5′ position of the 5′-terminalnucleotide.

Modified sugars also include sugar mimetics such as cyclobutyl orcyclopentyl moieties in place of the pentofuranosyl sugar.Representative United States patents that teach the preparation of suchmodified sugar structures include, but are not limited to, U.S. Pat.Nos. 4,981,957; 5,118,800; 5,319,080 ; and 5,359,044. Some modifiedsugars that are contemplated include

Other non-limiting examples of modified sugars include glycerol, whichform glycerol nucleic acid (GNA) analogues. One example of a GNAanalogue is shown below and is described in Zhang, R et al., J. Am.Chem. Soc., 2008, 130, 5846-5847; Zhang L, et al., J. Am. Chem. Soc.,2005, 127, 4174-4175 and Tsai C H et al., PNAS, 2007, 14598-14603:

wherein X is as defined herein. Another example of a GNA derivedanalogue, flexible nucleic acid (FNA) based on the mixed acetal aminalof formyl glycerol, is described in Joyce G F et al., PNAS, 1987, 84,4398-4402 and Heuberger B D and Switzer C, J. Am. Chem. Soc., 2008, 130,412-413, and is shown below:

Blocking Groups

In the reactions described, it is necessary in certain embodiments toprotect reactive functional groups, for example hydroxy, amino, thiol orcarboxy groups, where these are desired in the final product, to avoidtheir unwanted participation in the reactions. Protecting groups areused to block some or all reactive moieties and prevent such groups fromparticipating in chemical reactions until the protective group isremoved. In one embodiment, each protective group is removable by adifferent means. Protective groups that are cleaved under totallydisparate reaction conditions fulfill the requirement of differentialremoval. In some embodiments, protective groups are removed by acid,base, and/or hydrogenolysis. Groups such as trityl, dimethoxytrityl,acetal and t-butyldimethylsilyl are acid labile and are used in certainembodiments to protect carboxy and hydroxy reactive moieties in thepresence of amino groups protected with Cbz groups, which are removableby hydrogenolysis, and/or Fmoc groups, which are base labile. In otherembodiments, carboxylic acid and hydroxy reactive moieties are blockedwith base labile groups such as, but not limited to, methyl, ethyl, andacetyl in the presence of amines blocked with acid labile groups such ast-butylcarbamate or with carbamates that are both acid and base stablebut hydrolytically removable.

In another embodiment, hydroxy reactive moieties are blocked withhydrolytically removable protective groups such as the benzyl group,while amine groups capable of hydrogen bonding with acids are blockedwith base labile groups such as Fmoc. In another embodiment, carboxylicacid reactive moieties are protected by conversion to simple estercompounds, or they are, in yet another embodiment, blocked withoxidatively-removable protective groups such as 2,4-dimethoxybenzyl,while co-existing amino groups are blocked with fluoride labile silyl orcarbamate blocking groups.

Allyl blocking groups are useful in the presence of acid- andbase-protecting groups since the former are stable and can besubsequently removed by metal or pi-acid catalysts. For example, anallyl-blocked hydroxy groups can be deprotected with a Pd(0)-catalyzedreaction in the presence of acid labile t-butylcarbamate or base-labileacetate amine protecting groups. Yet another form of protecting group isa resin to which a compound or intermediate is attached. As long as theresidue is attached to the resin, that functional group is blocked andcannot react. Once released from the resin, the functional group isavailable to react.

Typically blocking/protecting groups are, by way of example only:

Representative protecting groups useful to protect nucleotides duringsynthesis include base labile protecting groups and acid labileprotecting groups. Base labile protecting groups are used to protect theexocyclic amino groups of the heterocyclic nucleobases. This type ofprotection is generally achieved by acylation. Three commonly usedacylating groups for this purpose are benzoyl chloride, phenoxyaceticanhydride, and isobutyryl chloride. These protecting groups are stableto the reaction conditions used during nucleic acid synthesis and arecleaved at approximately equal rates during the base treatment at theend of synthesis.

In some embodiments, the 5′-protecting group is trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 2-chlorotrityl, DATE, TBTr,9-phenylxanthine-9-yl (Pixyl), or 9-(p-methoxyphenyl)xanthine-9-yl(MOX).

In some embodiments, thiol moieties are incorporated in the compounds ofFormula 1, 2, 4, or 5 and are protected. In some embodiments, theprotecting groups include, but are not limited to, Pixyl, trityl,benzyl, p-methoxybenzyl (PMB), or tert-butyl (t-Bu).

Other protecting groups, plus a detailed description of techniquesapplicable to the creation of protecting groups and their removal aredescribed in Greene and Wuts, Protective Groups in Organic Synthesis,3rd Ed., John Wiley & Sons, New York, N.Y., 1999, and Kocienski,Protective Groups, Thieme Verlag, New York, N.Y., 1994, which areincorporated herein by reference for such disclosure.

In some embodiments, R¹ is —OR^(a), wherein R^(a) is substituted orunsubstituted trityl or substituted silyl. In other embodiments, R¹ is—N₃, —NR^(d)R^(d), alkynyloxy, or —OH. In some embodiments, R² is—OR^(b), wherein R^(b) is substituted or unsubstituted trityl,substituted silyl, acetyl, acyl, or substituted methyl ether. In otherembodiments, R² is —NR^(d)R^(d), alkyl, alkenyl, alkynyl, alkyl-Y¹—,alkenyl-Y¹—, alkynyl-Y¹—, aryl-Y¹—, heteroaryl-Y¹—, where Y¹ is O,NR^(d), S, or Se, and is substituted with fluorescent or biomoleculebinding moieties. In yet other embodiments, the substituent on R² is afluorescent moiety. In some embodiments, the substituent on R² is biotinor avidin. In some embodiments, R² is —OH, —N₃, hydrogen, halogen,alkoxy, or alkynyloxy.

In other embodiments, R³ is a blocking group which is substitutedtrityl, acyl, substituted silyl, or substituted benzyl. In yet otherembodiments, R³ is a linking moiety connected to a solid support. Infurther embodiments, the blocking group of the Ba moiety is a benzyl,acyl, formyl, dialkylformamidinyl, isobutyryl, phenoxyacetyl, or tritylmoiety, any of which may be unsubstituted or substituted.

Methods of Use of the Nucleic Acids Comprising a Chiral X-PhosphonateMoiety

The stereodefined oligonucleotides comprising a chiral X-phosphonatemoiety which are obtained by the methods of the invention are useful ina number of areas for applications due to a combination of stability,defined chirality and ease of synthesis. Broadly, the compoundssynthesized by this method are useful as therapeutics, diagnostic probesand reagents, synthetic tools for producing other oligonucleotideproducts, and nanostructure materials suitable for a variety of newmaterials and computing applications.

The stereodefined oligonucleotides of the invention have improved serumstability over that of natural DNA/RNA equivalents, and in particular,stereodefined oligonucleotides of the class of phosphorothioates.Further, the S_(P) isomer is more stable than the R_(P) isomer. In someembodiments, the level of serum stability is modulated by theintroduction of either all S_(P) centers or S_(P) centers at selectedpositions to confer resistance to degradation. In other embodiments,introduction of selectable R_(P) and/or S_(P) stereocenters can providefor specific base pairing association with an endogenous or exogenoustarget thus protecting the target from metabolism or enhancing aparticular biological reaction.

RNase H activation is also modulated by the presence of thestereocontrolled phosphorothioate nucleic acid analogs, with naturalDNA/RNA being more susceptible than the R_(P) stereoisomer which in turnis more susceptible than the corresponding S_(P) isomer.

Improved duplex stability towards RNA is seen with R_(P)phosphorothioate oligonucleotides having greater duplex stability thancorresponding S_(P) oligonucleotides which in turn demonstrates higherstability than that of natural DNA/RNA. Improved duplex stabilitytowards DNA is seen with S_(P) having greater duplex stability thanR_(P) which has more stability than that of natural DNA/RNA. (P. Guga,Curr. Top Med. Chem., 2007, 7, 695-713).

These molecules may be useful as therapeutic agents, in a number ofparticular applications. They can be incorporated into oligonucleotideswhich also contain the standard DNA/RNA nucleosides, or they may besynthesized as entire sequences of the stereocontrolled oligonucleotidesof the invention. Some categories of therapeutic agents include but arenot limited to antisense oligonucleotides, antigene oligonucleotideswhich form triple helix with targeted sequences to repress transcriptionof undesired genes and modulate protein expression and/or activity,decoy oligonucleotides, DNA vaccines, aptamers, ribozymes,deoxyribozymes (DNAzymes or DNA enzymes), siRNAs, microRNAs, ncRNAs(non-coding RNAs), and P-modified prodrugs. Modulation encompassesindirectly or directly increasing or decreasing the activity of aprotein or inhibition or promotion of the expression of a protein. Thesenucleic acid compounds can be used to control cell proliferation, viralreplication, or any other cell signaling process.

In one example, the field of siRNA therapeutics has a need foroligonucleotide species that can afford increased stability againstRNase activity, in order to improve the duration of action over thatseen with siRNA composed of natural nucleosides. Additionally, A-formhelix formation appears to be more indicative of success at enteringRNAi than the presence of specific native elements on theoligonucleotide. Both of these requirements can be afforded by the useof the stereocontrolled oligonucleotides of the invention may provideenhanced stability (Y-L Chiu, T. M. Rana RNA, 2003, 9,1034-1048).

The nucleic acids described herein are useful as therapeutic agentsagainst various disease states, including use as antiviral agents. Thenucleic acids can be used as agents for treatment of diseases throughmodulation of DNA and/or RNA activity. In some embodiments, the nucleicacids can be used for inhibiting specific gene expression. For example,the nucleic acids can be complementary to a specific target messengerRNA (mRNA) sequence. They can be used to inhibit viral replication suchas the orthopoxvirus, vaccinia virus, herpes, papilloma, influenza,cytomegalovirus and other viruses. Other examples include uses asantisense compounds against HIV RNA or other retroviral RNA or forhybridizing to HIV mRNA encoding the tat protein, or to the TAR regionof HIV mRNA. In some embodiments, the nucleic acids mimic the secondarystructure of the TAR region of HIV mRNA, and by doing so bind the tatprotein. In an embodiment, the nucleic acids is used to inhibitexpression of a target protein by contacting a cell with a compound ofFormula 1 wherein the expression of other proteins in the cell are notinhibited or are minimally inhibited. In some embodiment, target proteininhibition occurs in vivo in a mammal. In other embodiments, atherapeutically effective amount of a compound of Formula 1 isadministered for inhibiting the expression of a target protein.

Other examples of proteins where expression can be modulated include JunN-terminal kinase (INK) proteins, diacylglycerol acyltransferase I,apolipoprotein B, glucagon receptor, Aurora B, acyl CoA cholesterolacyltransferase-2, c-reactive protein, STAT (signal transducers andactivators of transcription) family of proteins, and MDR P-glycoprotein.The nucleic acids can be used to inhibit protein phosphatase 1B (PTP1B)expression, RNA-dependent RNA viral polymerase. The nucleic acids can beused to induce events such as apoptosis in cancer cells or to make acell more susceptible to apoptosis. The nucleic acids can be used tomodulate activities of proteins. For example, it can help modulate RNaseH activity on multidrug resistance (MDR) RNA molecules.

The nucleic acids described herein are useful for treating indicationsincluding, but not limited, to hypercholesterolemia, severe acuterespiratory syndrome (SARS), retroviral diseases such as AIDS or HIV,other viral infections, intrauterine infections, and cancer.

When used as therapeutics, the nucleic acid described herein isadministered as a pharmaceutical composition. In some embodiments, thepharmaceutical composition comprises a therapeutically effective amountof a nucleic acid comprising a chiral X-phosphonate moiety of Formula 1,or a pharmaceutically acceptable salt thereof, and at least onepharmaceutically acceptable inactive ingredient selected frompharmaceutically acceptable diluents, pharmaceutically acceptableexcipients, and pharmaceutically acceptable carriers. In anotherembodiment, the pharmaceutical composition is formulated for intravenousinjection, oral administration, buccal administration, inhalation, nasaladministration, topical administration, ophthalmic administration orotic administration. In further embodiments, the pharmaceuticalcomposition is a tablet, a pill, a capsule, a liquid, an inhalant, anasal spray solution, a suppository, a suspension, a gel, a colloid, adispersion, a suspension, a solution, an emulsion, an ointment, alotion, an eye drop or an ear drop.

A second category where the compounds synthesized by the methods of theinvention are useful are as primers or probes. Since the method providesfor total control of sequence, natural and unnatural, and ofstereochemistry at the phosphorus center, any specific molecule can bespecifically produced. Additionally, the additional RNase resistanceprovides molecules which are robust under ex-vivo or in-vivo conditions.The stereodefined oligonucleotides of the invention can be used asprobes for investigation of enzymatic reaction mechanisms involvingphosphorous atoms such as digestion, ligation, and polymerization ofnucleic acids. This class of molecules can be used as probes forinvestigation of ribozyme and deoxyribozyme reaction mechanisms. Theycan also function as probes for investigation of RNAi and othernon-coding RNA mediated gene silencing mechanisms or as probes foranalysis of protein-nucleic acid interactions. The ability to define thethree dimensional structure by incorporating select R_(P) or S_(P)phosphorus stereocenters permits the possibility of designing novelclasses of so-called molecular beacons.

As this method of synthesis is not limited to the narrow set of naturalnucleobases, modified nucleobases or other modifications to the base orsugar or termini permits the use of this class of oligonucleotides asprobes or sensors for nucleic acids, proteins and any biological orchemical substances in solution. They may be used similarly, withoutmodified nucleobases, using standard detection methods in place ofnatural DNA/RNA as well. Any of these may be incorporated as part of adiagnostic assay.

Such diagnostic tests can be performed using biological fluids, tissues,intact cells or isolated cellular components. As with gene expressioninhibition, diagnostic applications utilize the ability of the nucleicacids to hybridize with a complementary strand of nucleic acid.Hybridization is the sequence specific hydrogen bonding of oligomericcompounds via Watson-Crick and/or Hoogsteen base pairs to RNA or DNA.The bases of such base pairs are said to be complementary to oneanother. The nucleic acids can be used to analyze bodily states e.g.diseased states in animals. They can be used for identification ofadenoviruses or influenza viruses in a sample or as intercalating agentsor probes. For example, they can be used to detect DNA methylation orprobe DNA interactions with other cellular components such as proteins.

In another aspect of the invention, a method is provided of identifyingor detecting a target molecule in a sample, the method comprising:contacting a sample suspected of containing a target molecule with anucleic acid sensor molecule of Formula 1, synthesized according to themethods of the invention, wherein a change in a signal generated by asignal generating unit indicates the presence of said target in saidsample. The nucleic acid sensor molecule binds specifically with thetarget molecule. In some embodiments there is a plurality of nucleicacid sensor molecules. In some embodiments, the plurality of nucleicacid sensor molecules comprises nucleic acid sensor molecules which bindspecifically to differing target molecules. In some instances, themethod further comprises quantifying the change in signal generated bythe signal generating unit to quantify the amount of target molecule inthe sample. The signal generating unit detects any sort of signal,including but not limited to fluorescence, surface plasmon resonance,fluorescence quenching, chemiluminescence, interferometry, or refractiveindex detection.

The sample to be detected is an environmental sample, biohazardmaterial, organic sample, drug, toxin, flavor, fragrance, or biologicalsample. The biological sample is a cell, cell extract, cell lysate,tissue, tissue extract, bodily fluid, serum, blood or blood product. Insome embodiments of the method, the presence of the target moleculeindicates the presence of a pathological condition. In some embodimentsof the method, the presence of the target molecule indicates thepresence of a desirable molecule.

In a related use, the stereodefined oligonucleotides provided by themethods of the invention are useful as primers for PCR or as templatesor primers for DNA/RNA synthesis using polymerases. The meltingtemperatures may be optimized for a particular application depending onthe select introduction of R_(P) or S_(P) chirality at phosphorus in theproduct oligonucleotide.

In another aspect of the invention, a method is provided of amplifyingdesired regions of nucleic acid from a nucleic acid template comprising:(a) providing a plurality of first PCR primers having a region of fixednucleotide sequence complementary to a consensus sequence of interest;(b) providing a plurality of second PCR primers, (c) amplifying thenucleic acid template via the PCR using the plurality of first PCRprimers and the plurality of second PCR primers under conditions whereina subset of the plurality of first primers binds to the consensussequence of interest substantially wherever it occurs in the template,and a subset of the plurality of second primers binds to the template atlocations removed from the first primers such that nucleic acid regionsflanked by the first primer and the second primer are specificallyamplified, and wherein the plurality of first PCR primers and/or theplurality of second PCT primers are nucleic acid molecules of Formula 1which are produced according to the methods of the invention.

In some embodiments, the template is genomic DNA. In some embodiments,the template is eukaryotic genomic DNA. In some embodiments, thetemplate is human genomic DNA. In some embodiments, the template isprokaryotic DNA. In some embodiments, the template is DNA which is acloned genomic DNA, a subgenomic region of DNA, a chromosome, or asubchromosomal region. In some embodiments, the template is RNA.

The stereodefined oligonucleotides are also useful, due to theirincreased stability and their ability to retain recognition and bindingwith their biological targets, as substances for DNA chips andoligonucleotide microarrays. They may also be used as stabilizedoligonucleotides alternative to natural nucleic acids such as tRNA andmRNA in the cell free protein synthesis.

An additional area where the ability to control stability, molecularcomposition including unnatural moieties, and structure all within thesame synthesis is useful is for applications within DNA nanomaterialdesign. The stereodefined oligonucleotides of the invention may be usedas substances for construction of nucleic acid nano-structuresconsisting of duplex, triplex, quadruplex, and other higher-orderstructures. The ability to incorporate other organic moieties in themolecules produced by this methods leads to applications innanomaterials by designing a specific, unnatural higher order structure.The stability in-vivo and flexibility of design will permit thesemolecules' use in DNA computers, for example. Additionally, metalchelating or conducting organic molecules can be incorporated in thestereodefined oligonucleotides of the invention and lead to their use asDNA nano-wires in electronic devices or DNA/RNA nano-machines (F. A.Aldate, A. L. Palmer, Science, 2008, 321, 1795-1799).

EXAMPLES General Information:

All NMR spectra herein were recorded on a Varian Mercury 300. ¹H NMRspectra were obtained at 300 MHz with tetramethylsilane (TMS) (δ 0.0) asan internal standard in CDCl₃. ³¹P NMR spectra were obtained at 121.5MHz with 85% H₃PO₄ (δ 0.0) as an external standard. MALDI TOF-MS wererecorded on an Applied Biosystems Voyager System 4327. Silica gel columnchromatography was carried out using Kanto silica gel 60N (spherical,neutral, 63-210 μm). Analytical TLC was performed on Merck Kieselgel60-F₂₅₄ plates. Dry organic solvents were prepared by appropriateprocedures prior to use. The other organic solvents were reagent gradeand used as received. ACQUITY UPLC® was carried out using a BEH C₁₈ (1.7μm, 2.1×150 mm). The yield of the dTT, dCT, dAT, dGT, rU_(OMe)U, andrU_(F)U phosphorothioate dimers were determined by UV absorbancemeasurements at 260 nm with the molar extinction coefficients ofapproximate values for natural dTT (16800), dCT (15200), dAT (22800),dGT (20000), rUU (19600), rUU (19600) dimers, respectively.

Abbreviations:

bz: benzoyl

Beaucage reagent: 3H-1,2-benzodithiol-3-one 1,1-dioxide

BSA: N,O-bis(trimethylsilyl)acetamide

BTC: bis(trichloromethyl)carbonate

ce: cyanoethyl

CF₃COIm: N-trifluoroacetylimidazole

CMP: N-cyanomethyl pyrrolidine

CMPT: N-cyanomethyl pyrrolidinium trifluoromethanesulfonate

CPG: controlled pore glass

DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene

DCA: dichloroacetic acid

DCM: dichloromethane, CH₂Cl₂

DMAc: N,N-dimethylacetamide

DMAN: 1,8-bis(dimethylamino)naphthalene

DMTr: 4,4′-dimethoxytrityl

DTD: N,N′-dimethylthiuram disulfide

HCP: highly cross-linked polystyrene

pac: phenoxyacetyl

TBS: t-butyldimethylsilyl

TBDPS: t-butyldiphenylsilyl

TFA: trifluoroacetic acid

L-2: same as Formula P herein

D-2: same as Formula O herein

L-6: same as Formula R herein

D-6: same as Formula Q herein

Example 1 Solution Synthesis of a Phosphorothioate Dimer,(S_(P))-1,8-Diazabicyclo[5.4.0]undec-7-eniumN³-benzoyl-5′-O-(tert-butyldiphenylsilyl)thymidin-3′-ylN³-benzoyl-3′-O-(tert-butyldimethylsilyl)thymidin-5′-yl phosphorothioate[(S_(P))-4tt] via Route A

8-Diazabicyclo[5.4.0]undec-7-eniumN³-benzoyl-5′-O-(tert-butyldiphenylsilyl)thymidin-3′-yl phosphonate (1t)(96.0 mg, 120 μmol) was dried by repeated coevaporations with drypyridine and then dissolved in dry pyridine (2 mL). BTC (29.7 mg 100μmol) was added, and the mixture was stirred for 10 min. An aminoalcoholL-2 (21.3 mg, 120 μmol) solution, which was prepared by repeatedlycoevaporations with dry pyridine and dissolved in dry pyridine (1 mL),was added to the reaction mixture dropwise via syringe, and the mixturewas stirred for 5 min under argon atmosphere. To the solution ofN³-benzoyl-3′-O-(tert-butyldimethylsilyl)thymidine (3t), which wasprepared by repeated coevaporations with dry pyridine and dissolved inpyridine (500 μmol), the reaction mixture was added via syringe. After30 min, DTD (42.5 mg, 120 μmol) was added to the reaction mixture.Following an additional 5 min, the solvent was evaporated under thereduced pressure. Concentrated NH₃-EtOH (40 mL; 3:1, v/v) was added tothe residue, and the mixture was stirred for 12 h at room temperature.The mixture was concentrated to dryness under the reduced pressure. Thecrude mixture was diluted with CHCl₃ (15 mL), and washed with 0.2 Mtriethylammonium hydrogencarbonate (20 mL). The aqueous layer wasback-extracted with CHCl₃ (4×15 mL). The combined organic layers weredried over Na₂SO₄, filtered, and concentrated to dryness under thereduced pressure. The residue was purified by PTLC. The product wasdissolved in CHCl₃ (5 mL), washed with 0.2 M1,8-diazabicyclo[5.4.0]undec-7-enium hydrogencarbonate buffer (10 mL)and back-extracted with CHCl₃ (3×5 mL). The combined organic layers wasdried over Na₂SO₄, filtered, and concentrated to dryness to afford(S_(P))-4tt (41.8 mg, 98% yield, R_(P):S_(P)=9:91) as a white foam. The¹H and ³¹P NMR spectra (FIGS. 1 and 2, respectively) were identical tothose of a control sample synthesized by the conventional H-phosphonatemethod. ³¹P NMR (121.5 MHz, CDCl₃) δ 57.4 (R_(P) isomer: 57.9). Thesynthetic scheme is shown in Scheme 10.

Example 2 Solution Synthesis of a Phosphorothioate Dimer,(S_(P))-1,8-Diazabicyclo[5.4.0]undec-7-enium 6-N-benzoyl-5′-O-(tert-butyldiphenylsilyl)-deoxyadenosin-3′-yl3′-O-(tert-butyldimethylsilyl)thymidin-5′-yl phosphorothioate[(S_(P))-4at] via Route A

(S_(P))-4at is obtained from 1,8-diazabicyclo[5.4.0]undec-7-enium6-N-benzoyl-5′-O-(tert-butyldiphenylsilyl)-deoxyadenosin-3′-ylphosphonate (1a) instead of 1t, using the reaction steps described abovefor (S_(P))-4tt.

Example 3 Solution Synthesis of a Phosphorothioate Dimer,(S_(P))-1,8-Diazabicyclo[5.4.0]undec-7-enium4-N-benzoyl-5′-O-(tert-butyldiphenylsilyl)-deoxycytidin-3′-yl3′-O-(tert-butyldimethylsilyl)thymidin-5′-yl phosphorothioate[(S_(P))-4ct] via Route A

(S_(P))-4ct is obtained from 1,8-diazabicyclo[5.4.0]undec-7-enium4-N-benzoyl-5′-O-(tert-butyldiphenylsilyl)-deoxycytidin-3′-ylphosphonate (1c), instead of 1t, using the reaction steps describedabove for (S_(P))-4tt.

Example 4 Solution Synthesis of a Phosphorothioate Dimer,(S_(P))-1,8-Diazabicyclo[5.4.0]undec-7-enium2-N-phenoxyacetyl-5′-O-(tert-butyldiphenylsilyl)deoxyguanosin-3′-yl3′-O-(tert-butyldimethylsilyl)thymidin-5′-yl phosphorothioate[(S_(P))-4gt] via Route A

(S_(P))-4gt is obtained from 1,8-diazabicyclo[5.4.0]undec-7-enium2-N-phenoxyacetyl-5′-O-(tert-butyldiphenylsilyl)deoxyguanosin-3′-ylphosphonate (1g) instead of 1t, using the reaction steps described abovefor (S_(P))-4tt.

Example 5 Solution Synthesis of a Phosphorothioate Dimer,(R_(P))-1,8-Diazabicyclo[5.4.0]undec-7-eniumN³-benzoyl-5′-O-(tert-butyldiphenylsilyl)thymidin-3′-ylN³-benzoyl-3′-O-(tert-butyldimethylsilyl)thymidin-5′-yl phosphorothioate[(R_(P))-4tt] via Route A

(R_(P))-4tt was obtained as a white foam (93% yield, R_(P):S_(P)=95:5)by using amino alcohol D-2 instead of L-2 in a similar manner to(S_(P))-4tt in Example 1. The ¹H and ³¹P NMR spectra are shown in FIGS.3 and 4, respectively. ³¹P NMR (121.5 MHz, CDCl₃) δ 57.6 (S_(P) isomer:57.3.

Example 6 Solution Synthesis of a Phosphorothioate Dimer,(R_(P))-1,8-Diazabicyclo[5.4.0]undec-7-enium6-N-benzoyl-5′-O-(tert-butyldiphenylsilyl)deoxyadeno sin-3′-yl3′-O-(tert-butyldimethylsilyl)thymidin-5′-yl phosphorothioate[(R_(P))-4at] via Route A

(R_(P))-4at is produced via the transformations described above inExample 2 using compound 1a and the amino alcohol D-2 as a chiralreagent, instead of L-2.

Example 7 Solution Synthesis of a Phosphorothioate Dimer,(R_(P))-1,8-Diazabicyclo[5.4.0]undec-7-enium4-N-benzoyl-5′-O-(tert-butyldiphenylsilyl)deoxycytidin-3′-yl3′-O-(tert-butyldimethylsilyl)thymidin-5′-yl phosphorothioate[(R_(P))-4ct] via Route A

(R_(P))-4ct is produced via the transformations described above inExample 3 using compound 1c and the amino alcohol D-2 as a chiralreagent, instead of L-2.

Example 8 Solution Synthesis of a Phosphorothioate Dimer,(R_(P))-1,8-Diazabicyclo[5.4.0]undec-7-enium2-N-phenoxyacetyl-5′-O-(tert-butyldiphenylsilyl)deoxyguanosin-3′-yl3′-O-(tert-butyldimethylsilyl)thymidin-5′-yl phosphorothioate[(R_(P))-4gt] via Route A

(R_(P))-4gt is produced via the transformations described above inExample 4 using compound 1g and the amino alcohol D-2 as a chiralreagent, instead of L-2.

Example 9 Deprotection to form (S_(P))-Ammonium thymidin-3′-ylthymidin-5′-yl phosphorothioate [(S_(P))-5tt]

(S_(P))-4tt (50 μmol) is dried by repeated coevaporations with drypyridine and dry toluene, and then dissolved in triethylaminetrihydrofluoride (500 μL). The mixture is stirred for 15 h at roomtemperature. A 0.1 M ammonium acetate buffer (2.5 mL) is then added tothe mixture, and the mixture is washed with Et₂O (3×3 mL). The combinedorganic layers are back-extracted with 0.1 M ammonium acetate buffer (3mL). The combined aqueous layers are then concentrated to dryness underreduced pressure, and the residue is purified by reverse-phase columnchromatography [a linear gradient of acetonitrile 0-10% in 0.1 Mammonium acetate buffer (pH 7.0)] to afford (S_(P))-5tt. Thedeprotection scheme is shown in Scheme 11.

Example 10 Deprotection to form (S_(P))-Ammonium deoxyadenosin-3′-ylthymidin-5′-yl phosphorothioate [(S_(P))-5at]

(S_(P))-5at is produced as described above in Example 9 using(S_(P))-4at instead of (S_(P))-4tt.

Example 11 Deprotection to form (S_(P))-Ammonium deoxycytidin-3′-ylthymidin-5′-yl phosphorothioate [(S_(P))-5ct]

(S_(P))-5ct is produced as described above in Example 9 using(S_(P))-4ct instead of (S_(P))-4tt.

Example 12 Deprotection to form (S_(P))-Ammonium deoxyguanosin-3′-ylthymidin-5′-yl phosphorothioate [(S_(P))-5gt]

(S_(P))-5gt is produced as described above in Example 9 using(S_(P))-4gt instead of (S_(P))-4tt in.

Example 13 Deprotection to form (R_(P))-Ammonium thymidin-3′-ylthymidin-5′-yl phosphorothioate [(R_(P))-5tt]

(R_(P))-5tt is produced as described above in Example 9 using(R_(P))-4tt instead of (S_(P))-4tt in a similar manner as (S_(P))-5tt.

Example 14 Deprotection to form (R_(P))-Ammonium deoxyadenosin-3′-ylthymidin-5′-yl phosphorothioate [(R_(P))-5at]

(R_(P))-5at is produced as described above in Example 9 using(R_(P))-4at instead of (S_(P))-4tt.

Example 15 Deprotection to form (R_(P))-Ammonium deoxycytidin-3′-ylthymidin-5′-yl phosphorothioate [(R_(P))-5ct]

(R_(P))-5ct is produced as described above in Example 9 using(R_(P))-4ct instead of (S_(P))-4tt.

Example 16 Deprotection to form (R_(P))-Ammonium deoxyguanosin-3′-ylthymidin-5′-yl phosphorothioate [(R_(P))-5gt]

(R_(P))-5gt is produced as described above in Example 9 using(R_(P))-4gt instead of (S_(P))-4tt in a similar manner as (S_(P))-5tt.

Example 17 Synthesis of(R_(P))-5′-O-(tert-butyldiphenylsilyl)thymidin-3′-yl3′-O-(tert-butyldimethylsilyl)thymidin-5′-yl H-phosphonate [(R_(P))-7tt]via Route B

1t (100 μmol) is dried by repeated coevaporations with dry pyridine andthen dissolved in dry pyridine (1 mL).N,N′-Bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BopCl; 500 μmol) isadded, and the mixture is stirred for 5 min. To the mixture, a solutionof amino alcohol ((αR, 2S)-6) (100 μmol), which has been dried bycoevaportions with dry pyridine and dissolved in dry pyridine (1 mL), isadded dropwise via syringe, and the mixture is stirred for 5 min underargon. 3′-O-(tert-butyldimethylsilyl)thymidine is dried using repeatedcoevaporations with dry pyridine and dissolved in 100 μmol pyridine. Theabove mixture is added via cannula to the solution of3′-O-(tert-butyldimethylsilyl)thymidine 3t in dry (100 μmol) pyridine.After 15 min, the mixture is concentrated under reduced pressure. Theresidue is diluted with CH₂Cl₂ (5 mL), and washed with saturated NaHCO₃(3×5 mL). The combined aqueous layers are back-extracted with CH₂Cl₂(2×5 mL). The combined organic layers are dried over Na₂SO₄, filtered,and concentrated to ca. 1 mL under reduced pressure. The residue isadded dropwise via a syringe to a stirred 1% trifluoroacetic acid (TFA)solution in dry CH₂Cl₂ (20 mL) at 0° C. After an additional 5 min, themixture is diluted with dry CH₂Cl₂ (100 mL), and washed with saturatedNaHCO₃ aqueous solutions (2×100 mL). The combined aqueous layers areback-extracted with CH₂Cl₂ (2×100 mL). The combined organic layers aredried over Na₂SO₄, filtered, and concentrated to dryness under reducedpressure to afford crude (R_(P))-7tt. The synthetic scheme is shown inScheme 12.

Example 18 Synthesis of(R_(P))-6-N-benzoyl-5′-O-(tert-butyldiphenylsilyl)deoxyadenosin-3′-yl3′-O-(tert-butyldimethylsilyl)thymidin-5′-yl H-phosphonate [(R_(P))-7at]via Route B

Crude (R_(P))-7at is produced as described in Example 17 using 1ainstead of 1t.

Example 19 Synthesis of(R_(P))-4-N-benzoyl-5′-O-(tert-butyldiphenylsilyl)deoxycytidin-3′-yl3′-O-(tert-butyldimethylsilyl)thymidin-5′-yl H-phosphonate [(R_(P))-7ct]via Route B

Crude (R_(P))-7ct is produced as described in Example 17 using 1cinstead of 1t.

Example 20 Synthesis of(R_(P))-2-N-phenoxyacetyl-5′-O-(tert-butyldiphenylsilyl)deoxyguanosin-3′-yl3′-O-(tert-butyldimethylsilyl)thymidin-5′-yl H-phosphonate [(R_(P))-7gt]via Route B.

Crude (R_(P))-7gt is produced as described in Example 17 using 1ginstead of 1t.

Example 21 Synthesis of(S_(P))-5′-O-(tert-butyldiphenylsilyl)thymidin-3′-yl3′-O-(tert-butyldimethylsilyl)thymidin-5′-yl H-phosphonate [(S_(P))-7tt]via Route B

Crude (S_(P))-7tt is produced as described in Example 17 using (αS,2R)-6 instead of (αR, 2S)-6 as a chiral reagent.

Example 22 Synthesis of(S_(P))-6-N-benzoyl-5′-O-(tert-butyldiphenylsilyl)deoxyadenosin-3′-yl3′-O-(tert-butyldimethylsilyl)thymidin-5′-yl H-phosphonate [(S_(P))-7at]via Route B

Crude (S_(P))-7at is produced as described in Example 17 using compound1a and (αS, 2R)-6 instead of (αR, 2S)-6 as a chiral reagent.

Example 23 Synthesis of(S_(P))-4-N-benzoyl-5′-O-(tert-butyldiphenylsilyl)deoxycytidin-3′-yl3′-O-(tert-butyldimethylsilyl)thymidin-5′-yl H-phosphonate [(S_(P))-7ct]via Route B

Crude (S_(P))-7ct is produced as described in Example 17 using compound1c and (αS, 2R)-6 instead of (αR, 2S)-6 as a chiral reagent.

Example 24 Synthesis of(S_(P))-2-N-phenoxyacetyl-5′-O-(tert-butyldiphenylsilyl)deoxyguanosin-3′-yl3′-O-(tert-butyldimethylsilyl)thymidin-5′-yl H-phosphonate [(S_(P))-7gt]via Route B

Crude (S_(P))-7gt is produced as described in Example 17 using compound1g instead of 1t and compound (αS, 2R)-6 instead of compound (αR, 2S)-6as a chiral reagent.

Example 25 Modification to produce (R_(P))-Ammonium thymidin-3′-ylthymidin-5′-yl phosphorothioate [(R_(P))-5tt] as shown in general Scheme13

(S_(P))-7tt is dried by repeated coevaporations with dry pyridine anddry toluene, and then dissolved in CH₃CN (1 mL).N,O-bis(trimethylsilyl)acetamide (BSA; 100 μL) is added. After 1 min,N,N′-dimethylthiuram disulfide (DTD; 120 μmol) is added. After anadditional 3 min, the mixture is concentrated to dryness under reducedpressure to yield crude (R_(P))-4tt. Then the crude (R_(P))-4tt isdissolved in triethylamine trihydrofluoride (1 mL). The mixture isstirred for 15 h at room temperature. A 0.1 M ammonium acetate buffer (5mL) is then added to the mixture, and the mixture is washed with Et₂O(3×5 mL). The combined organic layers are back-extracted with 0.1 Mammonium acetate buffer (5 mL). The combined aqueous layers are thenconcentrated to dryness under reduced pressure, and the residue ispurified by reverse-phase column chromatography [a linear gradient ofacetonitrile 0-10% in 0.1 M ammonium acetate buffer (pH 7.0)] to afford(R_(P))-5tt. The modification steps are shown in Scheme 13.

Example 26 Modification to produce (R_(P))-Ammonium deoxyadenosin-3′-ylthymidin-5′-yl phosphorothioate [(R_(P))-5at]

(R_(P))-5at is produced as described in Example 25 using (S_(P))-7atinstead of (S_(P))-7tt.

Example 27 Modification to produce (R_(P))-Ammonium deoxycytidin-3′-ylthymidin-5′-yl phosphorothioate [(R_(P))-5ct]

(R_(P))-5ct is produced as described in Example 25 using (S_(P))-7ctinstead of (S_(P))-7tt.

Example 28 Modification to produce (R_(P))-Ammonium deoxyguanosin-3′-ylthymidin-5′-yl phosphorothioate [(R_(P))-5 gt]

(R_(P))-5gt is produced as described in Example 25 using (S_(P))-7gtinstead of (S_(P))-7tt.

Example 29 Modification to produce (S_(P))-Ammonium thymidin-3′-ylthymidin-5′-yl phosphorothioate [(S_(P))-5tt]

(S_(P))-5tt is produced as described in Example 25 using (R_(P))-7ttinstead of (S_(P))-7tt.

Example 30 Modification to produce (S_(P))-Ammonium deoxyadenosin-3′-ylthymidin-5′-yl phosphorothioate [(S_(P))-5at]

(S_(P))-5at is produced as described in Example 25 using (R_(P))-7atinstead of (S_(P))-7tt.

Example 31 Modification to produce (S_(P))-Ammonium deoxycytidin-3′-ylthymidin-5′-yl phosphorothioate [(S_(P))-5ct]

(S_(P))-5ct is produced as described in Example 25 using (R_(P))-7ctinstead of (S_(P))-7tt.

Example 32 Modification to produce (S_(P))-Ammonium deoxyguanosin-3′-ylthymidin-5′-yl phosphorothioate [(S_(P))-5gt]

(S_(P))-5gt is produced as described in Example 25 using (R_(P))-7gtinstead of (S_(P))-7tt.

Example 33 Synthesis of an RNA Analog Dimer,(S_(P))-1,8-Diazabicyclo[5.4.0]undec-7-enium5′-O-(tert-butyldiphenylsilyl)-2′-O-(tert-butyldimethylsilyl)uridin-3′-yl2′,3′-O-bis(tert-butyldimethylsilyl)uridin-5′-yl phosphorothioate[(S_(P))-10uu] via Route A

1,8-Diazabicyclo[5.4.0]undec-7-enium5′-O-(tert-butyldiphenylsilyl)-2′-O-(tert-butyldimethylsilyl)uridin-3′-ylphosphonate (8u) (100 μmol) is dried by repeated coevaporations with drypyridine and then dissolved in dry pyridine (1 mL).N,N′-Bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BopCl; 500 μmol) isadded, and the mixture is stirred for 5 min. To the mixture, a solutionof amino alcohol (L-2) (100 μmol), which has been dried by repeatedcoevaportions with dry pyridine and dissolved in dry pyridine (1 mL), isadded dropwise via syringe, and the mixture is stirred for 5 min underargon. 2′,3′-O-bis(tert-butyldimethylsilyl)uridine 9u is dried byrepeated coevaporations with dry pyridine and dissolved in 100 μmolpyridine. Then the above mixture is added via cannula into the solutionof 2′,3′-O-bis(tert-butyldimethylsilyl)uridine 9u (100 μmol). After 10min, N-trifluoroacetyl imidazole (CF₃COIm; 200 μmol) is added. After anadditional 30 s, N,N′-dimethylthiuram disulfide (DTD; 120 μmol) isadded. After an additional 3 min, the mixture is dried in vacuum. To theresidue, conc NH₃-EtOH (3:1, v/v, 10 mL) is added, and the mixture isstirred for 12 h, and then concentrated to dryness under reducedpressure. Then, the mixture is diluted with CHCl₃ (5 mL), and washedwith 0.2 M phosphate buffer (pH 7.0, 5 mL). The aqueous layers areback-extracted with CHCl₃ (2×5 mL). The combined organic layers aredried over Na₂SO₄, filtered, and concentrated to dryness under reducedpressure. The residue is purified by PTLC. The product is dissolved inCHCl₃ (5 mL), washed with 0.2 M 1,8-diazabicyclo[5.4.0]undec-7-eniumbicarbonate buffer (5 mL) and back-extracted with CHCl₃ (2×5 mL). Thecombined organic layers are dried over Na₂SO₄, filtered, andconcentrated to dryness to afford (S_(P))-10uu. The synthetic scheme isshown in Scheme 14.

Example 34 Synthesis of an RNA Analog Dimer,(S_(P))-1,8-Diazabicyclo[5.4.0]undec-7-enium6-N-benzoyl-5′-O-(tert-butyldiphenylsilyl)-2′-O-(tert-butyldimethylsilyl)adenosin-3′-yl2′,3′-O-bis(tert-butyldimethylsilyl)uridin-5′-yl phosphorothioate[(S_(P))-10au] via Route A

(S_(P))-10au is produced as described in Example 33 using1,8-diazabicyclo[5.4.0]undec-7-enium6-N-benzoyl-5′-O-(tert-butyldiphenylsilyl)-2′-O-(tert-butyldimethylsilyl)adenosin-3′-ylphosphonate (8a) instead of 8u

Example 35 Synthesis of an RNA Analog Dimer,(S_(P))-1,8-Diazabicyclo[5.4.0]undec-7-enium4-N-benzoyl-5′-O-(tert-butyldiphenylsilyl)-2′-O-(tert-butyldimethylsilyl)cytidin-3′-yl2′,3′-O-bis(tert-butyldimethylsilyl)uridin-5′-yl phosphorothioate[(S_(P))-10cu] via Route A

(S_(P))-10cu is produced as described in Example 33 using1,8-diazabicyclo[5.4.0]undec-7-enium4-N-benzoyl-5′-O-(tert-butyldiphenylsilyl)-2′-O-(tert-butyldimethylsilyl)cytidin-3′-ylphosphonate (8c) instead of 8u

Example 36 Synthesis of an RNA Analog Dimer,(S_(P))-1,8-Diazabicyclo[5.4.0]undec-7-enium2-N-phenoxyacetyl-5′-O-(tert-butyldiphenylsilyl)-2′-O-(tert-butyldimethylsilyl)guanosin-3′-yl2′,3′-O-bis(tert-butyldimethylsilyl)uridin-5′-yl phosphorothioate[(S_(P))-10gu] via Route A

(S_(P))-10gu is produced as described in Example 33 using1,8-diazabicyclo[5.4.0]undec-7-enium2-N-phenoxyacetyl-5′-O-(tert-butyldiphenylsilyl)-2′-O-(tert-butyldimethylsilyl)guanosin-3′-ylphosphonate (8g) instead of 8u.

Example 37 Synthesis of an RNA Analog Dimer,(R_(P))-1,8-Diazabicyclo[5.4.0]undec-7-enium5′-O-(tert-butyldiphenylsilyl)-2′-O-(tert-butyldimethylsilyl)uridin-3′-yl2′,3′-O-bis(tert-butyldimethylsilyl)uridin-5′-yl phosphorothioate[(R_(P))-10uu] via Route A

(R_(P))-10uu is produced as described in Example 33 using chiral reagentD-2 instead of chiral reagent L-2.

Example 38 Synthesis of an RNA Analog Dimer,(R_(P))-1,8-Diazabicyclo[5.4.0]undec-7-enium6-N-benzoyl-5′-O-(tert-butyldiphenylsilyl)-2′-O-(tert-butyldimethylsilyl)adenosin-3′-yl2′,3′-O-bis(tert-butyldimethylsilyl)uridin-5′-yl phosphorothioate[(R_(P))-10au] via Route A

(R_(P))-10au is produced as described in Example 33 using 8a instead of8u and chiral reagent D-2 instead of chiral reagent L-2.

Example 39 Synthesis of an RNA Analog Dimer,(R_(P))-1,8-Diazabicyclo[5.4.0]undec-7-enium4-N-benzoyl-5′-O-(tert-butyldiphenylsilyl)-2′-O-(tert-butyldimethylsilyl)cytidin-3′-yl2′,3′-O-bis(tert-butyldimethylsilyl)uridin-5′-yl phosphorothioate[(R_(P))-10cu] via Route A

(R_(P))-10cu is produced as described in Example 33 using 8c instead of8u and chiral reagent D-2 instead of chiral reagent L-2.

Example 40 Synthesis of an RNA Analog Dimer,(R_(P))-1,8-Diazabicyclo[5.4.0]undec-7-enium2-N-phenoxyacetyl-5′-O-(tert-butyldiphenylsilyl)-2′-O-(tert-butyldimethylsilyl)guanosin-3′-yl2′,3′-O-bis(tert-butyldimethylsilyl)uridin-5′-yl phosphorothioate[(R_(P))-10gu] via Route A

(R_(P))-10gu is produced as described in Example 33 using 8g instead of8u and chiral reagent D-2 instead of chiral reagent L-2.

Example 41 Deprotection to form (S_(P))-Triethylammonium uridin-3′-yluridin-5′-yl phosphorothioate [(S_(P))-11uu]

(S_(P))-10uu (50 μmol) is dried by repeated coevaporations with drypyridine and dry toluene, and then dissolved in 1M tetrabutylammoniumfluoride (TBAF) solution in dry THF (500 μL). The mixture is stirred for12 h at room temperature. A 0.05M triethylammonium acetate buffersolution (pH 6.9, 2.5 mL) is added to the mixture, and the mixture iswashed with Et₂O (3×3 mL). The combined organic layers areback-extracted with 0.05M triethylammonium acetate buffer (3 mL). Thecombined aqueous layers are then concentrated to dryness under reducedpressure, and the residue is purified by reverse-phase columnchromatography [a linear gradient of acetonitrile 0-10% in 0.1Mtriethylammonium acetate buffer (pH 6.9)] to afford (S_(P))-11uu. Thedeprotection scheme is shown in Scheme 15.

Example 42 Deprotection to form (S_(P))-Triethylammonium adenosin-3′-yluridin-5′-yl phosphorothioate [(S_(P))-11au]

(S_(P))-11au is produced as described in Example 41 using (S_(P))-10auinstead of (S_(P))-10uu.

Example 43 Deprotection to form (S_(P))-Triethylammonium cytidin-3′-yluridin-5′-yl phosphorothioate [(S_(P))-11 cu]

(S_(P))-11cu is produced as described in Example 41 using (S_(P))-10cuinstead of (S_(P))-10uu.

Example 44 Deprotection to form (S_(P))-Triethylammonium guanosin-3′-yluridin-5′-yl phosphorothioate [(S_(P))-11gu]

(S_(P))-11gu is produced as described in Example 41 using (S_(P))-10guinstead of (S_(P))-10uu.

Example 45 Deprotection to form (R_(P))-Triethylammonium uridin-3′-yluridin-5′-yl phosphorothioate [(R_(P))-11uu]

(R_(P))-11uu is produced as described in Example 41 using (R_(P))-10uuinstead of (S_(P))-10uu.

Example 46 Deprotection to form (R_(P))-Triethylammonium adenosin-3′-yluridin-5′-yl phosphorothioate [(R_(P))-11au]

(R_(P))-11au is produced as described in Example 41 using (R_(P))-10auinstead of (S_(P))-10uu.

Example 47 Deprotection to form (R_(P))-Triethylammonium cytidin-3′-yluridin-5′-yl phosphorothioate [(R_(P))-11cu]

(R_(P))-11cu is produced as described in Example 41 using (R_(P))-10cuinstead of (S_(P))-10uu.

Example 48 Deprotection to form (R_(P))-Triethylammonium guanosin-3′-yluridin-5′-yl phosphorothioate [(R_(P))-11gu]

(R_(P))-11gu is produced as described in Example 41 using (R_(P))-10guinstead of (S_(P))-10uu.

Example 49

Synthesis of an RNA Analog Dimer,(R_(P))-5′-O-(tert-butyldiphenylsilyl)-2′-O-(tert-butyldimethylsilyl)uridin-3′-yl2′,3′-O-bis(tert-butyldimethylsilyl)uridin-5′-yl H-phosphonate[(R_(P))-12uu] via Route B

8u (100 μmol) is dried by repeated coevaporations with dry pyridine andthen dissolved in dry pyridine (1 mL).N,N′-Bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BopCl; 500 μmol) isadded, and the mixture is stirred for 5 min. To the mixture, a solutionof amino alcohol ((αR, 2S)-6) (100 μmol), which is dried bycoevaportions with dry pyridine and dissolved in dry pyridine (1 mL), isadded dropwise via syringe, and the mixture is stirred for 5 min underargon. Then the mixture is added via cannula into a solution of 9u (100μmol), which is prepared by repeated coevaporations with dry pyridineand dissolution in pyridine. After 15 min, the mixture is concentratedunder reduced pressure. The residue is diluted with CH₂Cl₂ (5 mL), andwashed with saturated NaHCO₃ (3×5 mL). The combined aqueous layers areback-extracted with CH₂Cl₂ (2×5 mL). The combined organic layers aredried over Na₂SO₄, filtered, and concentrated to ca. 1 mL under reducedpressure. The residue is added dropwise via a syringe to a stirred 1%trifluoroacetic acid (TFA) solution in dry CH₂Cl₂ (20 mL) at 0° C. Afteran additional 5 min, the mixture is diluted with dry CH₂Cl₂ (100 mL),and washed with saturated NaHCO₃ aqueous solutions (2×100 mL). Thecombined aqueous layers are back-extracted with CH₂Cl₂ (2×100 mL). Thecombined organic layers are dried over Na₂SO₄, filtered, andconcentrated to dryness under reduced pressure to afford crude(R_(P))-12uu, which is analyzed by ³¹P NMR. The synthetic scheme isshown in Scheme 16.

Example 50 Synthesis of an RNA Analog Dimer,(R_(P))-6-N-Benzoyl-5′-O-(tert-butyldiphenylsilyl)-2′-O-(tert-butyldimethylsilyl)adenosin-3′-yl2′,3′-O-bis(tert-butyldimethylsilyl)uridin-5′-yl H-phosphonate[(R_(P))-12au] via Route B

Crude (R_(P))-12au is produced as described in Example 49 using 8ainstead of 8u.

Example 51 Synthesis of an RNA Analog Dimer,(R_(P))-4-N-Benzoyl-5′-O-(tert-butyldiphenylsilyl)-2′-O-(tert-butyldimethylsilyl)cytidin-3′-yl2′,3′-O-bis(tert-butyldimethylsilyl)uridin-5′-yl H-phosphonate[(R_(P))-12cu] via Route B

Crude (R_(P))-12cu is produced as described in Example 49 using 8cinstead of 8u.

Example 52 Synthesis of an RNA Analog Dimer,(R_(P))-2-N-Phenoxyacetyl-5′-O-(tert-butyldiphenylsilyl)-2′-O-(tert-butyldimethylsilyl)guanosin-3′-yl2′,3′-O-bis(tert-butyldimethylsilyl)uridin-5′-yl H-phosphonate[(R_(P))-12gu] via Route B

Crude (R_(P))-12gu is produced as described in Example 49 using 8ginstead of 8u.

Example 53 Synthesis of an RNA Analog Dimer,(S_(P))-5′-O-(tert-butyldiphenylsilyl)-2′-O-(tert-butyldimethylsilyl)uridin-3′-yl2′,3′-O-bis(tert-butyldimethylsilyl)uridin-5′-yl H-phosphonate[(S_(P))-12uu] via Route B

Crude (S_(P))-12uu is produced as described in Example 49 using chiralreagent (αS, 2R)-6 instead of chiral reagent (αR, 2S)-6.

Example 54 Synthesis of an RNA Analog Dimer,(S_(P))-6-N-Benzoyl-5′-O-(tert-butyldiphenylsilyl)-2′-O-(tert-butyldimethylsilyl)adenosin-3′-yl2′,3′-O-bis(tert-butyldimethylsilyl)uridin-5′-yl H-phosphonate[(S_(P))-12au] via Route B

Crude (S_(P))-12au is produced as described in Example 49 using 8ainstead of 8u and chiral reagent (αS, 2R)-6 instead of chiral reagent(αR, 2S)-6.

Example 55 Synthesis of an RNA Analog Dimer,(S_(P))-4-N-Benzoyl-5′-O-(tert-butyldiphenylsilyl)-2′-O-(tert-butyldimethylsilyl)cytidin-3′-yl2′,3′-O-bis(tert-butyldimethylsilyl)uridin-5′-yl H-phosphonate[(S_(P))-12cu] via Route B

Crude (S_(P))-12cu is produced as described in Example 49 using 8cinstead of 8u and chiral reagent (αS, 2R)-6 instead of chiral reagent(αR, 2S)-6.

Example 56 Synthesis of an RNA Analog Dimer,(S_(P))-2-N-Phenoxyacetyl-5′-O-(tert-butyldiphenylsilyl)-2′-O-(tert-butyldimethylsilyl)guanosin-3′-yl2′,3′-O-bis(tert-butyldimethylsilyl)uridin-5′-yl H-phosphonate[(S_(P))-12gu] via Route B

Crude (S_(P))-12gu is produced as described in Example 49 using 8ginstead of 8u and chiral reagent (αS, 2R)-6 instead of chiral reagent(αR, 2S)-6.

Example 57 Modification to Form (R_(P))-Triethylammonium uridin-3′-yluridin-5′-yl phosphorothioate [(R_(P))-11uu]

(S_(P))-12uu is dried by repeated coevaporation with dry pyridine anddry toluene, and then dissolved in CH₃CN (1 mL).N,O-bis(trimethylsilyl)acetamide (BSA; 100 μL) is added. After 1 min,N,N′-dimethylthiuram disulfide (DTD; 120 μmol) is added. After anadditional 3 min, the mixture is concentrated to dryness under reducedpressure to yield crude (R_(P))-10uu. Then the crude (R_(P))-10uu isdissolved in 1M tetrabutylammonium fluoride (TBAF) solution in dry THF(1 mL). The mixture is stirred for 12 h at room temperature. A 0.05Mtriethylammonium acetate buffer solution (pH 6.9, 5 mL) is added to themixture, and the mixture is washed with Et₂O (3×5 mL). The combinedorganic layers are back-extracted with 0.05M triethylammonium acetatebuffer (5 mL). The combined aqueous layers are then concentrated todryness under reduced pressure, and the residue is purified byreverse-phase column chromatography [a linear gradient of acetonitrile0-10% in 0.1M triethylammonium acetate buffer (pH 6.9)] to afford(R_(P))-11uu. The modification scheme is shown in Scheme 17.

Example 58 Modification to Form (R_(P))-Triethylammonium adenosin-3′-yluridin-5′-yl phosphorothioate [(R_(P))-11 au]

(R_(P))-11au is produced as described in Example 57 using (S_(P))-12auinstead of (S_(P))-12uu.

Example 59 Modification to Form (R_(P))-Triethylammonium cytidin-3′-yluridin-5′-yl phosphorothioate [(R_(P))-11cu]

(R_(P))-11cu is produced as described in Example 57 using (R_(P))-12cuinstead of (R_(P))-12uu.

Example 60 Modification to Form (R_(P))-Triethylammonium guanosin-3′-yluridin-5′-yl phosphorothioate [(R_(P))-11gu]

(R_(P))-11gu is produced as described in Example 57 using (S_(P))-12guinstead of (S_(P))-12uu.

Example 61 Modification to Form (S_(P))-Triethylammonium uridin-3′-yluridin-5′-yl phosphorothioate [(S_(P))-11uu]

(S_(P))-11uu is produced as described in Example 57 using (R_(P))-12uuinstead of (S_(P))-12uu.

Example 62 Modification to form (S_(P))-Triethylammonium adenosin-3′-yluridin-5′-yl phosphorothioate [(S_(P))-11au]

(S_(P))-11au is produced as described in Example 57 using (R_(P))-12auinstead of (S_(P))-12uu.

Example 63

Modification to Form (S_(P))-Triethylammonium cytidin-3′-yl uridin-5′-ylphosphorothioate [(S_(P))-11cu]

(S_(P))-11cu is produced as described in Example 57 using (R_(P))-12cuinstead of (S_(P))-12uu.

Example 64 Modification to Form (S_(P))-Triethylammonium guanosin-3′-yluridin-5′-yl phosphorothioate [(S_(P))-11gu]

(S_(P))-11gu is produced as described in Example 57 using (R_(P))-12guinstead of (S_(P))-12uu in a similar manner as (R_(P))-11uu.

Example 65 Solid Phase Synthesis of DNA Analogs Having X-PhosphonateMoieties Via Scheme 5 (Route A)

5′-O-(DMTr)thymidine-loaded HCP or CPG resin (0.5 μmol) via a succinyllinker is used for the synthesis. Chain elongation is performed byrepeating the steps in Table 1. After the chain elongation, the5′-O-DMTr group is removed by treatment with 3% DCA in CH₂Cl₂ (3×5 s),and washed with CH₂Cl₂. The oligomer on the HCP or CPG resin is thentreated with 25% NH₃-pyridine (9:1, v/v) for 15 h at 55° C. to removethe chiral auxiliaries and the protecting groups of the nucleobases andalso to release the oligomer from the HCP or CPG resin. The HCP or CPGresin is removed by filtration and washed with H₂O. The filtrate isconcentrated to dryness. The residue is dissolved in H₂O, washed withEt₂O, and the combined washings are back-extracted with H₂O. Thecombined aqueous layers are concentrated to dryness. The resulting crudeproduct is analyzed and/or purified by reversed-phase HPLC with a lineargradient of 0-20% acetonitrile in 0.1M ammonium acetate buffer (pH 7.0)for 60 min at 50° C. at a rate of 0.5 ml/min to afford stereoregularX-phosphonate DNAs.

TABLE 1 step Operation reagents and solvent time 1 detritylation 3% DCAin CH₂Cl₂ 3 × 30 s 2 washing (i) CH₂Cl₂ (ii) dry pyridine (iii) — dryingin vacuo. 3 coupling pre-activated monomer (0.2M)* 15 min in drypyridine 4 washing (i) dry pyridine (ii) dry CH₃CN — (iii) drying invacuo. 5 transformation sulfur electrophile, selenium 5 minelectrophile, or borane agent 6 washing (i) dry THF (ii) drying invacuo. — 7 capping CF₃COIm - 2,6-lutidine - dry 30 s THF (1:1:8, v/v/v)under argon 8 washing (i) dry THF (ii) CH₂Cl₂ —

* preparation of pre-activated monomer in Step 3 of Table 1:1,8-Diazabicyclo[5.4.0]undec-7-enium5′-O-(DMTr)-2′-deoxyribonucleoside-3′-yl phosphonate is dried byrepeated coevaporations with dry pyridine and then dissolved in drypyridine. BopCl is added to the solution, and the mixture is stirred for5 min. To the mixture, a solution of amino alcohol (L-2 or D-2), whichis dried by repeated coevaportions with dry pyridine and dissolved indry pyridine, is added dropwise via syringe, and the mixture is stirredfor 5 min under argon.

All-(R_(P))-[T_(PS)]₉T (Phosphorothioate).

According to the typical procedure described above, 5′-O-(DMTr)thymidine3′-O-succinate bound to HCP (0.5 μmol) give all-(R_(P))-[T_(PS)]₉T [1.52A₂₆₀ units, 17.7 nmol (35%) based on the assumption of 7%hypochoromicity: UV (H₂O) α_(max) 267 nm, α_(min) 236 nm] afterpurification of one-tenth of the crude product by RP-HPLC. <4% of thepurified oligomer is digested by incubation with nuclease P1 for 1 h at37° C.

Example 66 Solid Phase Synthesis of DNA Analogs Having X-PhosphonateMoieties Via Scheme 6 (Route B)

5′-O-(DMTr)thymidine-loaded CPG resin via a succinyl or oxalyl linker istreated 1% TFA in CH₂Cl₂ (3×5 s) for the removal of the 5′-O-DMTr group,washed with CH₂Cl₂ and dry pyridine and dried in vacuo. Chain elongationis performed by repeating the following steps (a) and (b). (a) Couplingreaction using a solution containing the corresponding pre-activatedmonomer* (0.2M) in dry pyridine (10 min) under argon. After thecondensation, the solid-support is washed with dry pyridine and CH₂Cl₂.(b) Removal of the 5 ′-O-DMTr group and the chiral auxiliarysimultaneously by treatment with 1% TFA in CH₂Cl₂Et₃SiH (1:1, v/v) (3×5s), and following washings with CH₂Cl₂ and dry pyridine. The resultantoligonucleoside H-phosphonates on the resin are converted toX-phosphonate DNAs as described below.

* preparation of pre-activated monomer:1,8-Diazabicyclo[5.4.0]undec-7-enium5′-O-(DMTr)-2′-deoxyribonucleoside-3′-yl phosphonate is dried byrepeated coevaporations with dry pyridine and then dissolved in drypyridine. BopCl is added to the solution, and the mixture is stirred for5 min. To the mixture, a solution of amino alcohol (L-6 or D-6), whichis dried by repeated coevaportion with dry pyridine and dissolved in drypyridine, is added dropwise via syringe, and the mixture is stirred for5 min under argon.

Phosphorothioate (X═S⁻).

Oligonucleoside H-phosphonate loaded to a CPG resin via a succinyllinker obtained as above is treated with 10 wt % S₈ inCS₂pyridinetriethylamine (35:35:1, v/v/v) at RT for 3 h, andsuccessively washed with CS₂, pyridine, and CH₃CN. The resin is treatedwith a 25% NH₃ aqueous solution at RT over 12 h, and washed with H₂O.The aqueous solutions are combined and concentrated to dryness underreduced pressure, and the residue is purified by RP-HPLC to affordstereoregulated phosphorothioate DNAs.

Boranophosphate (X═BH₃ ⁻).

Dry DMF, N,O-bis(trimethylsilyl)acetamide (BSA), and BH₃.SMe₂ are addedto the oligonucleside H-phosphonate loaded to a CPG resin via a oxalyllinker obtained as above at RT. After 15 min, the resin is successivelywashed with DMF, CH₃CN, and CH₃OH. The resin is then treated with asaturated NH₃ solution in CH₃OH at RT for 12 h, and washed with CH₃OH.The CH₃OH solutions are combined and concentrated to dryness underreduced pressure, and the residue is purified by RP-HPLC to affordstereoregulated boranophosphate DNAs.

Hydroxymethyl phosphonate (X═CH₂OH).

Oligonucleoside H-phosphonate loaded to a CPG resin via a oxalyl linkerobtained as above is treated with 0.1 M trimethylsilylchloride (TMSCl)in pyridine-1-methyl-2-pyrrolidone (NMP) (1:9, v/v) at RT for 10 min,and with gaseous formaldehyde at RT for 30 min, and then washed withNMP, and CH₃CN. The resin is then treated with a 25% NH₃ aqueoussolution at RT for 12 h, and washed with H₂O. The combined aqueoussolutions are concentrated to dryness under reduced pressure, and theresidue is purified by RP-HPLC to afford stereoregulated hydroxymethylphosphonate DNAs.

Phosphoramidate (X═NH₂).

Oligonucleoside H-phosphonate loaded to a CPG resin via an oxalyl linkerobtained as above is treated with a saturated NH₃ solution inCCl₄-1,4-dioxane (4:1, v/v) at 0° C. for 30 min, and washed with1,4-dioxane. The combined organic solutions are concentrated to drynessunder reduced pressure, treated with a 25% NH₃ aqueous solution at RTfor 12 h, and washed with H₂O. The combined aqueous solutions areconcentrated to dryness under reduced pressure, and the residue ispurified by RP-HPLC to afford stereoregulated phosphoramidate DNAs.

N-propyl phosphoramidate (X═NHPr).

Oligonucleoside H-phosphonate loaded to a CPG resin via a oxalyl linkerobtained as above is treated with CCl₄-propylamine (9:1, v/v) at RT for1 h, and washed with CH₃OH. The combined organic solutions areconcentrated to dryness under reduced pressure, treated with a 25% NH₃aqueous solution at RT for 12 h, and washed with H₂O. The combinedaqueous solutions are concentrated to dryness under reduced pressure,and the residue is purified by RP-HPLC to afford stereoregulatedN-propylphophoramidate DNAs. N-[(2-dimethylamino)ethyl]phosphoramidate[X═NH(CH₂)₂NMe₂].

Oligonucleoside H-phosphonate loaded to a CPG resin via a oxalyl linkerobtained as above is treated with CCl₄-2-dimethylaminoethylamine (9:1,v/v) at RT for 1 h, and washed with CH₃CN. The combined organicsolutions are concentrated to dryness under reduced pressure, treatedwith a 25% NH₃ aqueous solution at RT for 12 h, and washed with H₂O. Thecombined aqueous solutions are concentrated to dryness under reducedpressure, and the residue is purified by RP-HPLC to affordstereoregulated N-[(2-dimethylamino)ethyl]phosphoramidate DNAs.

Example 67 Synthesis of All-(S_(P))-d[C_(S)A_(S)G_(S)T](Phosphorothioate)

The corresponding oligonucleoside H-phosphonate is synthesized on a CPGresin via a succinyl linker as described above, and treated with a 0.2Msolution of Beaucage reagent in BSA-CH₃CN (1:8, v/v) at RT for 30 min,and the resin is washed with CH₃CN. The resin is then treated with a 25%NH₃ aqueous solution at RT for 12 h, and washed with H₂O. The combinedaqueous solutions are concentrated to dryness under reduced pressure,and the residue is analyzed and characterized by RP-HPLC andMALDI-TOF-MS. The RP-HPLC is performed with a linear gradient of 0-20%acetonitrile in 0.1M ammonium acetate buffer (pH 7.0) for 60 min at 50°C. at a flow rate of 0.5 mL/min using a μBondasphere 5 μm C18 column(100 Å, 3.9 mm×150 mm) (Waters).

Example 68 Synthesis of All-(R_(P))-d[C_(S)A_(S)G_(S)T](Phosphorothioate)

The corresponding oligonucleoside H-phosphonate is synthesized on a CPGresin via a succinyl linker as described above, and treated with a 0.2Msolution of Beaucage reagent in BSA-CH₃CN (1:8, v/v) (0.2 mL) at RT for30 min, and the resin is washed with CH₃CN. The resin is treated with a25% NH₃ aqueous solution (5 mL) at RT for 12 h, and washed with H₂O. Thecombined aqueous solutions are concentrated to dryness under reducedpressure, and the residue is analyzed and characterized by RP-HPLC andMALDI-TOF-MS. The RP-HPLC is performed with a linear gradient of 0-20%acetonitrile in 0.1 M ammonium acetate buffer (pH 7.0) for 60 min at 50°C. at a flow rate of 0.5 mi./min using a μBondasphere 5 μm C18 column(100 Å, 3.9 mm×150 mm) (Waters).

Example 69 Synthesis of All-(S_(P))-[T_(S)]₉T (Phosphorothioate)

The corresponding oligonucleoside H-phosphonate is synthesized on a CPGresin via a succinyl linker as described above, and treated with a 0.2Msolution of Beaucage reagent in BSA-CH₃CN (1:8, v/v) (0.2 mL) at RT for30 min, and the resin is washed with CH₃CN. The CPG resin is treatedwith a 25% NH₃ aqueous solution (5 mL) at RT for 24 h, and washed withH₂O. The combined aqueous solutions are concentrated to dryness underreduced pressure, and the residue is analyzed and characterized byRP-HPLC and MALDI-TOF-MS. The RP-HPLC is performed with a lineargradient of 0-20% acetonitrile in 0.1M ammonium acetate buffer (pH 7.0)for 80 min at 30° C. at a flow rate of 0.5 mL/min using a μBondasphere 5μm C18 column (100 Å, 3.9 mm×150 mm) (Waters).

Example 70 Synthesis of All (R_(P))-[T_(S)]₉T (Phosphorothioate)

The corresponding oligonucleoside H-phosphonate is synthesized on a CPGresin via a succinyl linker as described above, and treated with a 0.2Msolution of Beaucage reagent in BSA-CH₃CN (1:8, v/v) (0.2 mL) at RT for30 min, and the resin is washed with CH₃CN. The resin is treated with a25% NH₃ aqueous solution (5 mL) at RT for 24 h, and washed with H₂O. Thecombined aqueous solutions are concentrated to dryness under reducedpressure, and the residue is analyzed and characterized by RP-HPLC andMALDI-TOF-MS. The RP-HPLC is performed with a linear gradient of 0-20%acetonitrile in 0.1M ammonium acetate buffer (pH 7.0) for 80 min at 30°C. at a flow rate of 0.5 mL/min using a μBondasphere 5 μm C18 column(100 Å, 3.9 mm×150 mm) (Waters).

Example 71 Synthesis of Al (R_(P))-[T_(B)]₃T (Boranophosphate)

The corresponding oligonucleoside H-phosphonate is synthesized on a CPGresin via a succinyl linker as described above, and treated with amixture of (dry DMF (0.8 mL), BSA (0.1 mL) and BH₃S(CH₃)₂ (0.1 mL) at RTfor 15 min, and the resin is successively washed with DMF, CH₃CN, andCH₃OH. The resin is then treated with a saturated solution of NH₃ inCH₃OH (5 mL) at RT for 2 h, and washed with CH₃OH. The combined organicsolutions are concentrated to dryness under reduced pressure, and theresidue is analyzed and characterized by RP-HPLC and MALDI-TOF-MS. TheRP-HPLC is performed with a linear gradient of 0-20% acetonitrile in0.1M ammonium acetate buffer (pH 7.0) for 60 min at 30° C. at a flowrate of 0.5 ml/min using a PEGASIL ODS 5 μm (120 Å, 4.0 mm×150 mm)(Senshu Pak).

Example 72 Synthesis of All-(S_(P))-[T_(B)]₃T (Boranophosphate)

The corresponding oligonucleoside H-phosphonate is synthesized on a CPGresin via a succinyl linker as described above, and treated with amixture of dry DMF (0.8 mL), BSA (0.1 mL) and BH₃S(CH₃)₂ (0.1 mL) at RTfor 15 min, and the resin is successively washed with DMF, CH₃CN, andCH₃OH. The resin is then treated with a saturated solution of NH₃ inCH₃OH (5 mL) at RT for 2 h, and washed with CH₃OH. The combined organicsolutions are concentrated to dryness under reduced pressure, and theresidue is analyzed and characterized by RP-HPLC and MALDI-TOF-MS. TheRP-HPLC is performed with a linear gradient of 0-20% acetonitrile in0.1M ammonium acetate buffer (pH 7.0) for 60 min at 30° C. at a flowrate of 0.5 mL/min using a PEGASIL ODS 5 μm (120 Å, 4.0 mm×150 mm)(Senshu Pak).

Example 73 Synthesis of All-(S_(P))-[T_(N)]₃T(N-[(2-dimethylamino)ethyl]phosphoramidate)

The corresponding oligonucleoside H-phosphonate is synthesized on a CPGresin via an oxalyl linker as described above, and treated withCCl₄-2-dimethylaminoethylamine (9:1, v/v) at RT for 1 h, and washed withCH₃CN. The combined organic solutions are concentrated to dryness underreduced pressure, and the residue is analyzed and characterized byRP-HPLC and MALDI-TOF-MS. The RP-HPLC is performed with a lineargradient of 0-20% acetonitrile in 0.1M triethylammonium acetate buffer(pH 7.0) for 60 min at 30° C. at a flow rate of 0.5 mL/min using aPEGASIL ODS 5 μm (120 Å, 4 0 mm×150 mm) (Senshu Pak).

Example 74 Synthesis of All-(R_(P))-[T_(N)]₃T(N-[(2-dimethylamino)ethyl]phosphoramidate)

The corresponding oligonucleoside H-phosphonate is synthesized on a CPGresin via an oxalyl linker as described above, and treated withCCl₄-2-dimethylaminoethylamine (9:1, v/v) at RT for 1 h, and washed withCH₃CN. The combined organic solutions are concentrated to dryness underreduced pressure, and the residue is analyzed and characterized byRP-HPLC and MALDI-TOF-MS. The RP-HPLC is performed with a lineargradient of 0-20% acetonitrile in 0.1M triethylammonium acetate buffer(pH 7.0) for 60 min at 30° C. at a flow rate of 0.5 mL/min using aPEGASIL ODS 5 μm (120 Å, 4.0 mm×150 mm) (Senshu Pak).

Example 75 A General Procedure for Solid-Phase Synthesis ofX-Phosphonate RNA Via Scheme 5 (Route A)

5′-O-(DMTr)uridine-loaded HCP or CPG resin via a succinyl linker is usedfor the synthesis. Chain elongation is performed by repeating the stepsin Table 2. After the chain elongation, the 5′-O-DMTr group is removedby treatment with 3% DCA in CH₂Cl₂ and the resin is successively washedwith CH₂Cl₂ and EtOH. The resin is then treated with a 25% NH₃ aqueoussolutionEtOH (3:1, v/v) for 2 h at room temperature and removed byfiltration. The filtrate is diluted with a 25% NH₃ aqueous solutionEtOH(3:1, v/v) and placed in a tightly-sealed flask for 48 h at roomtemperature. The solution is concentrated under reduced pressure, andthe residue is purified by RP-HPLC. Fractions containing the desired2′-O-TBS-protected X-phosphonate RNAs are collected and lyophilized. Theresidue is treated with a 1M TBAF solution in dry THF for 24 h at roomtemperature. A 0.05M TEAA buffer solution (pH 6.9) is added, and THF isremoved by evaporation. The residue is desalted with a Sep-pak C₁₈cartridge, and purified by RP-HPLC to afford stereoregular X-phosphonateRNAs.

TABLE 2 step Operation reagents and solvent time 1 detritylation 3% DCAin CH₂Cl₂ 4 × 30 s 2 washing (i) CH₂Cl₂ (ii) dry pyridine (iii) — dryingin vacuo. 3 coupling pre-activated monomer (0.2M)* 15 min in drypyridine 4 washing (i) dry pyridine (ii) dry CH₃CN — (iii) drying invacuo. 5 transformation sulfur electrophile, selenium 5 minelectrophile, or borane agent 6 washing (i) dry THF (ii) drying invacuo. — 7 capping CF₃COIm - 2,6-lutidine - dry 30 s THF (1:1:8, v/v/v)under argon 8 washing (i) dry THF (ii) CH₂Cl₂ —

* preparation of pre-activated monomer in Step 3 of Table 2:1,8-Diazabicyclo[5.4.0]undec-7-enium5′-O-(DMTr)-2′-O-(TBS)-ribonucleoside-3′-yl phosphonate is dried byrepeated coevaporations with dry pyridine and then dissolved in drypyridine. BopCl is added to the solution, and the mixture is stirred for5 min. To the mixture, a solution of amino alcohol (L-2 or D-2), whichis repeated coevaportions with dry pyridine and dissolved in drypyridine, is added dropwise via syringe, and the mixture is stirred for5 min under argon.

Example 76 A General Procedure for Solid-Phase Synthesis ofX-Phosphonate RNA via Scheme 6 (Route B)

Procedure to synthesis H-phosphonate RNA. 5′-O-(DMTr)uridine-loaded CPGresin via a succinyl or oxalyl linker is treated 1% TFA in CH₂Cl₂ (3×5s) for the removal of the 5′-O-DMTr group, washed with CH₂Cl₂ and drypyridine and dried in vacuo. Chain elongation is performed by repeatingthe following steps (a) and (b). (a) Coupling reaction using a solutioncontaining the corresponding pre-activated monomer* (0.2M) in drypyridine (10 min) under argon. After the condensation, the solid-supportis washed with dry pyridine and CH₂Cl₂. (b) Removal of the 5′-O-DMTrgroup and the chiral auxiliary simultaneously by treatment with 1% TFAin CH₂Cl₂Et₃SiH (1:1, v/v) (3×5 s), and following washings with CH₂Cl₂and dry pyridine. The resultant oligonucleoside H-phosphonates on theresin are converted to backbone-modified RNA analogues as describedbelow.

* preparation of pre-activated monomer

1,8-Diazabicyclo[5.4.0]undec-7-enium5′-O-(DMTr)-2′-O-(TBS)-ribonucleoside-3′-yl phosphonate is dried byrepeated coevaporations with dry pyridine and then dissolved in drypyridine. BopCl is added to the solution, and the mixture is stirred for5 min. To the mixture, a solution of amino alcohol (L-6 or D-6), whichis dried by repeated coevaportions with dry pyridine and dissolved indry pyridine, is added dropwise via syringe, and the mixture is stirredfor 5 min under argon.

Phosphorothioate (X═S⁻).

Oligonucleoside H-phosphonate loaded to a CPG resin via a succinyllinker obtained as above is treated with 10 wt % S₈ inCS₂pyridinetriethylamine (35:35:1, v/v/v) at RT for 3 h, andsuccessively washed with CS₂, pyridine, and EtOH. The resin is thentreated with a 25% NH₃ aqueous solutionEtOH (3:1, v/v) for 2 h at roomtemperature and removed by filtration. The filtrate is diluted with a25% NH₃ aqueous solution-EtOH (3:1, v/v) and placed in a tightly-sealedflask for 12 h at room temperature. The solution is concentrated underreduced pressure, and the residue is purified by RP-HPLC. Fractionscontaining the desired 2′-O-TBS-protected phosphorothioate RNAs arecollected and lyophilized. The residue is treated with a 1M TBAFsolution in dry THF for 24 h at room temperature. A 0.05M TEAA buffersolution (pH 6.9) is added, and THF is removed by evaporation. Theresidue is desalted with a Sep-pak C₁₈ cartridge, and purified byRP-HPLC to afford stereoregular phosphorothioate RNAs.

Boranophosphate (X═BH₃ ⁻).

Dry DMF, N,O-bis(trimethylsilyl)acetamide (BSA), and BH₃SMe₂ are addedto the oligonucleside H-phosphonate loaded to a CPG resin via a oxalyllinker obtained as above at RT. After 15 min, the resin is successivelywashed with DMF, CH₃CN, and EtOH. The resin is then treated with a 25%NH₃ aqueous solution EtOH (3:1, v/v) for 2 h at room temperature andremoved by filtration. The filtrate is diluted with a 25% NH₃ aqueoussolutionEtOH (3:1, v/v) and placed in a tightly-sealed flask for 12 h atroom temperature. The solution is concentrated under reduced pressure,and the residue is purified by RP-HPLC. Fractions containing the desired2′-O-TBS-protected boranophosphate RNAs are collected and lyophilized.The residue is treated with a 1M TBAF solution in dry THF for 24 h atroom temperature. A 0.05M TEAA buffer solution (pH 6.9) is added, andTHF is removed by evaporation. The residue is desalted with a Sep-pakC₁₈ cartridge, and purified by RP-HPLC to afford stereoregularboranophosphate RNAs.

Hydroxymethyl phosphonate (X═CH₂OH).

Oligonucleoside H-phosphonate loaded to a CPG resin via a oxalyl linkerobtained as above is treated with 0.1M trimethylsilylchloride (TMSCl) inpyridine-1-methyl-2-pyrrolidone (NMP) (1:9, v/v) at RT for 10 min, andwith gaseous formaldehyde at RT for 30 min, and then washed with NMP,and EtOH. The resin is then treated with a 25% NH₃ aqueous solutionEtOH(3:1, v/v) for 2 h at room temperature and removed by filtration. Thefiltrate is diluted with a 25% NH₃ aqueous solutionEtOH (3:1, v/v) andplaced in a tightly-sealed flask for 12 h at room temperature. Thesolution is concentrated under reduced pressure, and the residue ispurified by RP-HPLC. Fractions containing the desired 2′-O-TBS-protectedhydroxymethyl phosphonate RNAs are collected and lyophilized. Theresidue is treated with a 1M TBAF solution in dry THF for 24 h at roomtemperature. A 0.05M TEAA buffer solution (pH 6.9) is added, and THF isremoved by evaporation. The residue is desalted with a Sep-pak C₁₈cartridge, and purified by RP-HPLC to afford stereoregular hydroxymethylphosphonate RNAs.

Phosphoramidate (X═NH₂).

Oligonucleoside H-phosphonate loaded to a CPG resin via an oxalyl linkerobtained as above is treated with a saturated NH₃ solution inCCl₄-1,4-dioxane (4:1, v/v) at 0° C. for 30 min, and washed with1,4-dioxane. The combined organic solutions are concentrated to drynessunder reduced pressure. The filtrate is diluted with a 25% NH₃ aqueoussolutionEtOH (3:1, v/v) and placed in a tightly-sealed flask for 12 h atroom temperature. The solution is concentrated under reduced pressure,and the residue is purified by RP-HPLC. Fractions containing the desired2′-O-TBS-protected phosphoramidate RNAs are collected and lyophilized.The residue is treated with a 1M TBAF solution in dry THF for 24 h atroom temperature. A 0.05M TEAA buffer solution (pH 6.9) is added, andTHF is removed by evaporation. The residue is desalted with a Sep-pakC₁₈ cartridge, and purified by RP-HPLC to afford stereoregularphosphoramidate RNAs.

N-propyl phosphoramidate (X═NHPr).

Oligonucleoside H-phosphonate loaded to a CPG resin via an oxalyl linkerobtained as above is treated with CCl₄-propylamine (9:1, v/v) at RT for1 h, and washed with CH₃OH. The combined organic solutions areconcentrated to dryness under reduced pressure. The filtrate is dilutedwith a 25% NH₃ aqueous solution EtOH (3:1, v/v) and placed in atightly-sealed flask for 12 h at room temperature. The solution isconcentrated under reduced pressure, and the residue is purified byRP-HPLC. Fractions containing the desired 2′-O-TBS-protectedN-propylphophoramidate RNAs are collected and lyophilized. The residueis treated with a 1M TBAF solution in dry THF for 24 h at roomtemperature. A 0.05M TEAA buffer solution (pH 6.9) is added, and THF isremoved by evaporation. The residue is desalted with a Sep-pak C₁₈cartridge, and purified by RP-HPLC to afford stereoregularN-propylphophoramidate RNAs.

N-[(2-dimethylamino)ethyl]phosphoramidate [X═NH(CH₂)₂NMe₂].

Oligonucleoside H-phosphonate loaded to a CPG resin via an oxalyl linkerobtained as above is treated with CCI₄-2-dimethylaminoethylamine (9:1,v/v) at RT for 1 h, and washed with CH₃CN. The combined organicsolutions are concentrated to dryness under reduced pressure. Thefiltrate is diluted with a 25% NH₃ aqueous solution EtOH (3:1, v/v) andplaced in a tightly-sealed flask for 12 h at room temperature. Thesolution is concentrated under reduced pressure, and the residue ispurified by RP-HPLC. Fractions containing the desired 2′-O-TBS-protectedN-[(2-dimethylamino)ethyl]phosphoramidate RNAs are collected andlyophilized. The residue is treated with a 1 M TBAF solution in dry THFfor 24 h at room temperature. A 0.05M TEAA buffer solution (pH 6.9) isadded, and THF is removed by evaporation. The residue is desalted with aSep-pak C₁₈ cartridge, and purified by RP-HPLC to afford stereoregularN-[(2-dimethylamino)ethyl]phosphoramidate RNAs.

Example 77 Solid-Phase Synthesis; General Procedure for the Preparationof Pre-Activated Monomer Solution

Appropriate H-phosphonate monoester was dried by repeated coevaporationswith dry pyridine and dry toluene, then dissolved in dry solvent. To thesolution, condensing reagent was added dropwise, and stirred for 10 min.Aminoalcohol was then added and stirred for additional 10 min to givepre-activated monomer solution.

Example 78 Solid-Phase Synthesis of a Phosphorothioate Dirtier,(S_(P))-Ammonium thymidin-3′-yl thymidin-5′-yl phosphorothioate[(S_(P))-5tt] via Route A

N³-Benzoyl-5′-O-(4,4′-dimethoxytrityl)thymidine-loaded HCP resin (16.4mg; 30.5 μmol/g, 0.5 μmol) via a succinyl linker was treated with 3%DCA/DCM (3×1 mL), then washed with DCM (3×1 mL) and dry MeCN (3×1 mL).After the resin was dried under the reduced pressure (>5 min),pre-activated monomer solution (250 μL, 25 μmol; which consists of8-diazabicyclo[5.4.0]undec-7-eniumN³-benzoyl-5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphonate (25μmol, for H-phosphonate monoester), MeCN-pyridine (9:1, v/v, forsolvent), Ph₃PCl₂ (62.5 μmol, for condensing reagent), and L-2 (30 μmol,for aminoalcohol)) was added. Being stirred for 2 min, the reactionsolution was removed, and the resin was washed with MeCN (3×1 mL), anddried under the reduced pressure (>5 min). For the modification step,the resulting intermediate on the resin was sulfurized by treatment with0.3 M DTD/MeCN (500 μL, 150 μmol) for 5 min, the resin was then washedwith MeCN (3×1 mL) and DCM (3×1 mL). The 5′-O-DMTr group was removed bytreatment with 3% DCA/DCM (3×1 mL), and the resin was washed with DCM(3×1 mL). The phosphorothioate dimer on the resin was then treated with25% NH₃ (1 mL) for 12 h at 55° C. to remove the chiral auxiliary and theprotecting groups of the nucleobases and also to release the dimer fromthe resin. The resin was removed by filtration and washed with H₂O. Thefiltrate was concentrated to dryness. The residue was dissolved in H₂O(2 mL), washed with Et₂O (3×2 mL), and the combined washings wereback-extracted with H₂O (2 mL). The combined aqueous layers wereconcentrated to dryness. The resulting crude product was analyzed byreversed-phase UPLC® with a linear gradient of 0-20% MeOH in 0.1 Mammonium acetate buffer (pH 7.0) for 15 min at 55° C. at a rate of 0.4ml/min. The product was identical to that of a control samplesynthesized by the conventional H-phosphonate method. The yield of(S_(P))-5tt was 97% (R_(P):S_(P)=2:98). Retention time: 13.4 min((R_(P))-5tt: 12.3 min). The general scheme is shown in Scheme 18. TheUPLC profile is shown in FIG. 5A. In another synthesis of (S_(P))-5tt,BTC (20 μmol) was used in placed of Ph₃PCl₂ (62.5 μmol). The product wasidentical to that of a control sample synthesized by the conventionalH-phosphonate method. The yield of (S_(P))-5tt was 95% yield(R_(P):S_(P)=3:97). Retention time: 13.5 min ((R_(P))-5tt: 12.4 min) theUPLC profile is shown in FIG. 5B.

Example 79 Solid-Phase Synthesis of a Phosphorothioate Dimer,(S_(P))-Ammonium thymidin-3′-yl thymidin-5′-yl phosphorothioate[(S_(P))-5tt]

N³-Benzoyl-5′-O-(4,4′-dimethoxytrityl)thymidine-loaded HCP resin (16.4mg; 30.5 μmol/g, 0.5 μmol) via a succinyl linker was treated with 3%DCA/DCM (3×1 mL), then washed with DCM (3×1 mL) and dry MeCN (3×1 mL).After the resin was dried under the reduced pressure (>5 min),pre-activated monomer solution (200 μL, 25 μmol; which consists of8-diazabicyclo[5.4.0]undec-7-eniumN³-benzoyl-5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphonate (25μmol, for H-phosphonate monoester), MeCN-CMP (9:1, v/v, for solvent),Ph₃PCl₂ (62.5 for condensing reagent), and L-2 (30 μmol, foraminoalcohol)) was added followed by the addition of 5 M CMPT/MeCN (50μL, 250 μmol, for activating reagent). Being stirred for 10 min, thereaction solution was removed, and the resin was washed with MeCN (3×1mL), dried under the reduced pressure (>5 min). The resultingintermediate on the resin was sulfurized by treatment with 0.3 MDTD/MeCN (500 μL, 150 μmol) for 5 min, then the resin was washed withMeCN (3×1 mL) and DCM (3×1 mL). The 5′-O-DMTr group was removed bytreatment with 3% DCA/DCM (3×1 mL), and washed with DCM (3×1 mL). Thephosphorothioate dimer on the resin was then treated with 25% NH₃ (1 mL)for 12 h at 55° C. to remove the chiral auxiliary and the protectinggroups of the nucleobases and also to release the dimer from the resin.The resin was removed by filtration and washed with H₂O. The filtratewas concentrated to dryness. The residue was dissolved in H₂O (2 mL),washed with Et₂O (3×2 mL), and the combined washings were back-extractedwith H₂O (2 mL). The combined aqueous layers were concentrated todryness. The resulting crude product was analyzed by reversed-phase UPLCwith a linear gradient of 0-20% MeOH in 0.1 M ammonium acetate buffer(pH 7.0) for 15 min at 55° C. at a rate of 0.4 ml/min. The product wasidentical to that of a control sample synthesized by the conventionalH-phosphonate method. The yield of (S_(P))-5tt was 98% yield(R_(P):S_(P)=1:99). Retention time: 13.5 min ((R_(P))-5tt: 12.4 min).The UPLC profile is shown in FIG. 6A. This compound was also obtained byusing “BTC (16 μmol) and L-2 (26 μmol)” instead of “Ph₃PCl₂ (62.5 μmol)and L-2 (30 μmol)” in a similar manner described. The product wasidentical to that of a control sample synthesized by the conventionalH-phosphonate method. The yield of (S_(P))-5tt was 95% yield(R_(P):S_(P)=1:99). Retention time: 13.5 min ((R_(P))-5tt: 12.4 min).The UPLC profile is shown in FIG. 6B.

Example 80 Solid-Phase Synthesis of a Phosphorothioate Dimer,(R_(P))-Ammonium thymidin-3′-yl thymidin-5′-yl phosphorothioate[(R_(P))-5tt] via Route A

This compound was obtained by using “D-2 (30 μmol)” instead of “L-2 (30μmol)” in a similar manner to “Example 78”. The product was identical tothat of a control sample synthesized by the conventional H-phosphonatemethod. The yield of (R_(P))-5tt was 98% (R_(P):S_(P)=97:3). Retentiontime: 12.2 min ((S_(P))-5tt: 13.5 min). The UPLC profile is shown inFIG. 7A. In another synthesis of (R_(P))-5tt, BTC (20 μmol) was used inplaced of Ph₃PCl₂ (62.5 μmol). The product was identical to that of acontrol sample synthesized by the conventional H-phosphonate method. Theyield of (R_(P))-5tt was 97% (R_(P):S_(P)=95:5). Retention time: 12.3min ((S_(P))-5tt: 13.6 min). The UPLC profile is shown in FIG. 7B.

Example 81 Solid-Phase Synthesis of a Phosphorothioate Dimer,(R_(P))-Ammonium thymidin-3′-yl thymidin-5′-yl phosphorothioate[(R_(P))-5tt] via Route A

This compound was obtained by using “D-2 (30 μmol)” instead of “L-2 (30μmol)” in a similar manner to “Example 79”. The product was identical tothat of a control sample synthesized by the conventional H-phosphonatemethod. The yield of (R_(P))-5tt was 98% (R_(P):S_(P)=98:2). Retentiontime: 12.3 min ((S_(P))-5tt: 13.6 min). The UPLC profile is shown inFIG. 8.

Example 82 Solid-Phase Synthesis of a Phosphorothioate Dimer,(S_(P))-Ammonium 2′-deoxycytidin-3′-yl thymidin-5′-yl phosphorothioate[(S_(P))-5ct] via Route A

This compound was obtained by using “8-diazabicyclo[5.4.0]undec-7-enium4-N-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidin-3′-ylphosphonate (25 μmol)” instead of “8-diazabicyclo[5.4.0]undec-7-eniumN³-benzoyl-5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphonate (25μmol)” in a similar manner to Example 78. The product was identical tothat of a control sample synthesized by the conventional H-phosphonatemethod. The yield of (S_(P))-5ct was 98% yield (R_(P):S_(P)=3:97).Retention time: 9.7 min ((R_(P))-5ct: 8.7 min). The UPLC profile isshown in FIG. 9A. This compound was also obtained by using “BTC (16μmol) and L-2 (26 μmol)” instead of “Ph₃PCl₂ (62.5 μmol) and L-2 (30μmol)” as described. The product was identical to that of a controlsample synthesized by the conventional H-phosphonate method. The yieldof (S_(P))-5ct was 94% (R_(P):S_(P)=3:97). Retention time 9.7 min((R_(P))-5ct: 8.7 min). The UPLC profile is shown in FIG. 9B.

Example 83 Solid-Phase Synthesis of a Phosphorothioate Dimer,(R_(P))-Ammonium 2′-deoxycytidin-3′-yl thymidin-5′-yl phosphorothioate[(R_(P))-5ct] via Route A

This compound was obtained by using “D-2 (30 μmol)” instead of “L-2 (30μmol)” in a similar manner to the experiment for Example 82, FIG. 9A.The product was identical to that of a control sample synthesized by theconventional H-phosphonate method. The yield of (R_(P))-5ct was 98%(R_(P):S_(P)=97:3). Retention time: 8.6 min ((S_(P))-5ct: 9.7 min). TheUPLC profile is shown in FIG. 10A. This compound was also obtained byusing “D-2 (30 μmol)” instead of “L-2 (30 μmol)” in a similar manner toExample 82, FIG. 9B. The yield of (R_(P))-5ct was 87%(R_(P):S_(P)=98:2). Retention time: 8.6 min ((S_(P))-5ct: 9.8 min). TheUPLC profile is shown in FIG. 10B.

Example 84 Solid-Phase Synthesis of a Phosphorothioate Dimer,(S_(P))-Ammonium 2′-deoxyadenin-3′-yl thymidin-5′-yl phosphorothioate[(S_(P))-5at] via Route A

This compound was obtained by using “8-diazabicyclo[5.4.0]undec-7-enium6-N,N-dibenzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenin-3′-ylphosphonate (25 μmol)” instead of “8-diazabicyclo[5.4.0]undec-7-eniumN³-benzoyl-5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphonate (25μmol)” in a similar manner to Example 78. The product was identical tothat of a control sample synthesized by the conventional H-phosphonatemethod. The yield of (S_(P))-5at was 96% yield (R_(P):S_(P)=1:99).Retention time: 14.0 min ((R_(P))-5at: 12.6 min). The UPLC profile isshown in FIG. 11.

Example 85 Solid-Phase Synthesis of a Phosphorothioate Dimer,(R_(P))-Ammonium 2′-deoxyadenin-3′-yl thymidin-5′-yl phosphorothioate[(R_(P))-5at] via Route A

This compound was obtained by using “D-2 (30 μmol)” instead of “L-2 (30μmol)” in a similar manner to Example 83. The product was identical tothat of a control sample synthesized by the conventional H-phosphonatemethod. The yield of (R_(P))-5at was 96% (R_(P):S_(P)=96:4). Retentiontime: 12.5 min ((S_(P))-5at: 14.1 min). The UPLC profile is shown inFIG. 12.

Example 86 Solid-Phase Synthesis of a Phosphorothioate Dimer,(S_(P))-Ammonium 2′-deoxyguanin-3′-yl thymidin-5′-yl phosphorothioate[(S_(P))-5gt] via Route A

This compound was obtained by using “8-diazabicyclo[5.4.0]undec-7-eniumO⁶-cyanoethyl-2-N-phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanin-3′-ylphosphonate (25 μmol)” instead of “8-diazabicyclo[5.4.0]undec-7-eniumN³-benzoyl-5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphonate (25μmol)” in a similar manner to Example 78. The product was identical tothat of a control sample sample synthesized by the conventionalH-phosphonate method. The yield of (S_(P))-5gt was 96%(R_(P):S_(P)=2:98). Retention time: 11.5 min ((R_(P))-5gt: 10.3 min).The UPLC profile is shown in FIG. 13.

Example 87 Solid-Phase Synthesis of a Phosphorothioate Dimer,(R_(P))-Ammonium 2′-deoxyguanin-3′-yl thymidin-5′-yl phosphorothioate[(R_(P))-5gt] via Route A

This compound was obtained by using “D-2 (30 μmol)” instead of “L-2 (30μmol)” in a similar manner to Example 86. The product was identical tothat of a control sample sample synthesized by the conventionalH-phosphonate method. The yield of (R_(P))-5gt was 96%(R_(P):S_(P)=97:3). Retention time: 10.3 min ((S_(P))-5gt: 11.6 min).The UPLC profile is shown in FIG. 14.

Example 88 Solid-Phase Synthesis of a Phosphorothioate Dimer,(S_(P))-Ammonium thymidin-3′-yl thymidin-5′-yl phosphorothioate[(S_(P))-5tt] via Route B

N³-Benzoyl-5′-O-(4,4′-dimethoxytrityl)thymidine-loaded HCP resin (16.4mg; 30.5 μmol/g, 0.5 μmol) via a succinyl linker was treated with 1%TFA/DCM (3×1 mL) for the removal of the 5′-O-DMTr group, washed with DCM(3×1 mL) and dry MeCN (3×1 mL), and dried in vacuo. Pre-activatedmonomer solution (200 μL, 50 μmol; which consists of8-diazabicyclo[5.4.0]undec-7-eniumN³-benzoyl-5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphonate (50μmol, for H-phosphonate monoester), MeCN-CMP (9:1, v/v, for solvent),Ph₃PCl₂ (125 μmol, for condensing reagent), and L-6 (52 μmol, foraminoalcohol)) was added followed by the addition of 5 M CMPT/MeCN (50μL, 250 μmol). Being stirred for 2 min, the reaction solution wasremoved, and the resin was washed with MeCN (3×1 mL), DCM (3×1 mL), anddried under the reduced pressure (>5 min). The 5′-O-DMTr group and thechiral auxiliary was simultaneously removed by treatment with 1% TFA inDCM (3×1 mL), washed with DCM (3×1 mL) and dry MeCN (3×1 mL), and driedin vacuo. The resulting intermediate on the resin was sulfurized bytreatment with the solution mixture of 0.2 M Beaucage reagent/MeCN (200μL, 40 μmol) and BSA (25 μl, 100 μmol) for 20 min, the resin was thenwashed with MeCN (3×1 mL). The phosphorothioate dimer on the resin wasthen treated with 25% NH₃-EtOH (2 mL, 4:1, v/v) for 12 h at roomtemperature to remove the protecting groups of the nucleobases and alsoto release the dimer from the resin. The resin was removed by filtrationand washed with H₂O. The filtrate was concentrated to dryness. Theresidue was dissolved in H₂O (2 mL), washed with Et₂O (3×2 mL), and thecombined washings were back-extracted with H₂O (2 mL). The combinedaqueous layers were concentrated to dryness. The resulting crude productwas analyzed by reversed-phase UPLC® with a linear gradient of 0-20%MeOH in 0.1 M ammonium acetate buffer (pH 7.0) for 15 min at 55° C. at arate of 0.4 ml/min. The product was identical to that of a controlsample synthesized by the conventional H-phosphonate method. The yieldof (S_(P))-5tt was 96% (R_(P):S_(P)=4:96). Retention time: 13.5 min((R_(P))-5tt: 12.4 min). The UPLC profile is shown in FIG. 15A.

In an alternative synthesis, this compound was also obtained by using“BTC (32 μmol)” instead of “Ph₃PCl₂ (125 μmol)” in a similar manner asdescribed. The product was identical to that of a control samplesynthesized by the conventional H-phosphonate method. The yield of(S_(P))-5tt was 96% (R_(P):S_(P)=5:95). Retention time: 13.4 min((R_(P))-5tt: 12.3 min). The UPLC profile is shown in FIG. 15B.

Example 89 Solid-Phase Synthesis of a Phosphorothioate Dimer,(S_(P))-Ammonium thymidin-3′-yl thymidin-5′-yl phosphorothioate[(S_(P))-5tt] via Route B

In another alternative synthesis, [(S_(P))-5tt] was obtained by using“8-diazabicyclo[5.4.0]undec-7-eniumN³-benzoyl-5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphonate (25μmol), BTC (16 μmol), and L-6 (26 μmol))” instead of“8-diazabicyclo[5.4.0]undec-7-eniumN³-benzoyl-5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphonate (50μmol), Ph₃PCl₂ (125 μmol), and L-6 (52 μmol)” in a similar manner toFIG. 15A Example 88. The general scheme is shown in Scheme 19. Theproduct was identical to that of a control sample synthesized by theconventional H-phosphonate method. The yield of (S_(P))-5tt was 93%(R_(P):S_(P)=6:94). Retention time: 13.5 min ((R_(P))-5tt: 12.4 min).The UPLC profile is shown in FIG. 16A. This compound was obtained byusing “0.2 M DTD/MeCN (200 μl, 80 μmol)” instead of “0.2 M Beaucagereagent/MeCN (200 μL, 40 μmol)” in a similar manner as described. Theproduct was identical to that of a control sample synthesized by theconventional H-phosphonate method. The yield of (S_(P))-5tt was 95%(R_(P):S_(P)=6:94). Retention time: 13.5 min ((R_(P))-5tt: 12.4 min).The UPLC profile is shown in FIG. 16B.

Example 90 Solid-Phase Synthesis of a Phosphorothioate Dimer,(R_(P))-Ammonium thymidin-3′-yl thymidin-5′-yl phosphorothioate[(R_(P))-5tt] via Route B

This compound was obtained by using “D-6 (52 μmol)” instead of “L-6 (52μmol)” in a similar manner to the methods in Example 88, FIG. 15A. Theproduct was identical to that of a control sample synthesized by theconventional H-phosphonate method. The yield of (R_(P))-5tt was 95%(R_(P):S_(P)=97:3). Retention time: 12.3 min ((S_(P))-5tt: 13.6 min).The UPLC profile is shown in FIG. 17A.

This compound was obtained by using “D-6 (52 μmol)” instead of “L-6 (52μmol)” in a similar manner to the methods in Example 88, FIG. 15B. Theproduct was identical to that of a control sample synthesized by theconventional H-phosphonate method. The yield of (R_(P))-5tt was 94%(R_(P):S_(P)=97:3). Retention time: 12 3 min ((S_(P))-5tt: 13.6 min).The UPLC profile is shown in FIG. 17B.

Example 91 Solid-Phase Synthesis of a Phosphorothioate Dimer,(S_(P))-Ammonium 2′-deoxycytidin-3′-yl thymidin-5′-yl phosphorothioate[(S_(P))-5ct] via Route B

This compound was obtained by using “8-diazabicyclo[5.4.0]undec-7-enium4-N-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidin-3′-ylphosphonate (50 μmol)” instead of “8-diazabicyclo[5.4.0]undec-7-eniumN³-benzoyl-5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphonate (50μmol)” in a similar manner to Example 88. The product was identical tothat of a control sample synthesized by the conventional H-phosphonatemethod. The yield of (S_(P))-5ct was 95% (R_(P):S_(P)=4:96). Retentiontime: 9.7 min ((R_(P))-5ct: 8.7 min). The UPLC profile is shown in FIG.18.

Example 92 Solid-Phase Synthesis of a Phosphorothioate Dimer,(R_(P))-Ammonium 2′-deoxycytidin-3′-yl thymidin-5′-yl phosphorothioate[(R_(P))-5ct] via Route B

This compound was obtained by using “D-6 (52 μmol)” instead of “L-6 (52μmol)” in a similar manner to Example 91. The product was identical tothat of a control sample synthesized by the conventional H-phosphonatemethod. The yield of (R_(P))-5ct was 96% (R_(P):S_(P)=97:3). Retentiontime: 8.6 min ((S_(P))-5ct: 9.8 min). The UPLC profile is shown in FIG.19.

Example 93 Solid-Phase Synthesis of a Phosphorothioate Dimer,(S_(P))-Ammonium 2′-deoxyadenin-3′-yl thymidin-5′-yl phosphorothioate[(S_(P))-5at] via Route B

This compound was obtained by using “8-diazabicyclo[5.4.0]undec-7-enium6-N,N-dibenzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenin-3′-ylphosphonate (50 μmol)” instead of “8-diazabicyclo[5.4.0]undec-7-eniumN³-benzoyl-5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphonate (50μmol)” in a similar manner to Example 88. The product was identical tothat of a control sample synthesized by the conventional H-phosphonatemethod. The yield of (S_(P))-5at was 95% yield, R_(P):S_(P)=5:95.Retention time: 14.0 min ((R_(P))-5at: 12.5 min). The UPLC profile isshown in FIG. 20A.

This compound was also obtained by using “BTC (32 μmol)” instead of“Ph₃PCl₂ (125 μmol)” in a similar manner to the method described in thisexample for FIG. 20A. The product was identical to that of a controlsample synthesized by the conventional H-phosphonate method. The yieldof (S_(P))-5at was 94% (R_(P):S_(P)=5:95). Retention time: 13.9 min((R_(P))-5at: 12.5 min). The UPLC profile is shown in FIG. 20B.

Example 94 Solid-Phase Synthesis of a Phosphorothioate Dimer,(S_(P))-Ammonium 2′-deoxyadenin-3′-yl thymidin-5′-yl phosphorothioate[(S_(P))-5at] via Route B

This compound was obtained by using “8-diazabicyclo[5.4.0]undec-7-enium6-N-((dimethylamino)methylene)-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenin-3′-ylphosphonate (50 μmol)” instead of “8-diazabicyclo[5.4.0]undec-7-eniumN³-benzoyl-5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphonate (50μmol)” in a similar manner to Example 88. The product was identical tothat of a control sample synthesized by the conventional H-phosphonatemethod. The yield of (S_(P))-5at was 91% yield, R_(P):S_(P)=5:95.Retention time: 13.9 min ((R_(P))-5at: 12.5 min). The UPLC profile isshown in FIG. 21.

Example 95 Solid-Phase Synthesis of a Phosphorothioate Dimer,(R_(P))-Ammonium 2′-deoxyadenin-3′-yl thymidin-5′-yl phosphorothioate[(R_(P))-5at] via Route B

This compound was obtained by using “D-6 (52 μmol)” instead of “L-6 (52μmol)” in a similar manner to the method for Example 93, FIG. 20A. Theproduct was identical to that of a control sample synthesized by theconventional H-phosphonate method. The yield of (R_(P))-5at was 96%yield, R_(P):S_(P)=97:3. Retention time: 12.5 min ((S_(P))-5at: 14.0min). The UPLC profile is shown in FIG. 22A.

This compound was obtained by using “D-6 (52 μmol)” instead of “L-6 (52μmol)” in a similar manner to the method for Example 93, FIG. 20B. Theproduct was identical to that of a control sample synthesized by theconventional H-phosphonate method. The yield of (R_(P))-5at was 94%(R_(P):S_(P)=95:5). Retention time: 12.4 min ((R_(P))-5at: 14.0 min).The UPLC profile is shown in FIG. 22B.

Example 96 Solid-Phase Synthesis of a Phosphorothioate Dimer,(S_(P))-Ammonium 2′-deoxyguanin-3′-yl thymidin-5′-yl phosphorothioate[(S_(P))-5gt] via Route B

This compound was obtained by using “8-diazabicyclo[5.4.0]undec-7-eniumO⁶-cyanoethyl-2-N-phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanin-3′-ylphosphonate (50 μmol)” instead of “8-diazabicyclo[5.4.0]undec-7-eniumN³-benzoyl-5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphonate (50μmol)” in a similar manner to Example 88. The product was identical tothat of a control sample synthesized by the conventional H-phosphonatemethod. The yield of (S_(P))-5gt was 95% (R_(P):S_(P)=6:94). Retentiontime: 11.5 min ((R_(P))-5gt: 10.3 min). The UPLC profile is shown inFIG. 23.

Example 97 Solid-Phase Synthesis of a Phosphorothioate Dimer,(R_(P))-Ammonium 2′-deoxyguanin-3′-yl thymidin-5′-yl phosphorothioate[(R_(P))-5gt] via Route B

This compound was obtained by using “D-2 (52 μmol)” instead of “L-2 (52μmol)” in a similar manner to Example 96. The product was identical tothat of a control sample synthesized by the conventional H-phosphonatemethod. The yield of (R_(P))-5gt was 95% (R_(P):S_(P)=94:6). Retentiontime: 10.3 min ((S_(P))-5gt: 11.6 min). The UPLC profile is shown inFIG. 24.

Example 98 Solid-Phase Synthesis of a Boranophoshate Dimer,(S_(P))-Ammonium thymidin-3′-yl thymidin-5′-yl boranophosphate[(S_(P))-7tt] via Route B

N³-Benzoyl-5′-O-(4,4′-dimethoxytrityl)thymidine-loaded HCP resin (16.4mg; 30.5 μmol/g, 0.5 μmol) via a succinyl linker was treated with 1%TFA/DCM (3×1 mL) for the removal of the 5′-O-DMTr group, washed with DCM(3×1 mL) and dry MeCN (3×1 mL), and dried in vacuo. Pre-activatedmonomer solution (200 μL, 50 μmol; which consists of8-diazabicyclo[5.4.0]undec-7-eniumN³-benzoyl-5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphonate (50μmol, for H-phosphonate monoester), MeCN-CMP (9:1, v/v, for solvent),BTC (32 μmol, for condensing reagent), and D-6 (52 μmol, foraminoalcohol)) was added followed by the addition of 5 M CMPT/MeCN (50μL, 250 μmol). Being stirred for 2 min, the reaction solution wasremoved, and the resin was washed with MeCN (3×1 mL), DCM (3×1 mL), anddried under the reduced pressure (>5 min). The 5′-O-DMTr group and thechiral auxiliary was simultaneously removed by treatment with 1% TFA inDCM (3×1 mL), and the resin was washed with DCM (3×1 mL) and dry MeCN(3×1 mL), and dried in vacuo. The resulting intermediate on the resinwas boronated by treatment with a mixture of BH₃SMe₂-BSA-DMAc (1 mL,1:1:8, v/v/v) for 15 min, the resin was then washed with DMAc (3×1 mL),MeCN (3×1 mL), and MeOH (3×1 mL). The boranophosphate dimer on the resinwas then treated with 2 M NH₃/EtOH (2 mL) for 12 h at room temperatureto remove the protecting groups of the nucleobases and also to releasethe dimer from the resin. The resin was removed by filtration and washedwith MeOH. The filtrate was concentrated to dryness. The residue wasdissolved in H₂O (2 mL), washed with Et₂O (3×2 mL), and the combinedwashings were back-extracted with H₂O (2 mL). The combined aqueouslayers were concentrated to dryness. The resulting crude product wasanalyzed by reversed-phase UPLC® with a linear gradient of 0-15% MeCN in0.1 M ammonium acetate buffer (pH 7.0) for 15 min at 60° C. at a rate of0.5 ml/min. The product was identical to that of a control samplesynthesized by the conventional H-phosphonate method. The yield of(S_(P))-7tt was determined by a UV absorbance measurement at 260 nm withthe molar extinction coefficient of an approximate value for natural TTdimer (16800). The yield of (S_(P))-7tt was 89% (R_(P):S_(P)=4:96).Retention time: 9.6 min ((R_(P))-7tt: 9.8 min). The UPLC profile isshown in FIG. 25.

Example 99 Solid-Phase Synthesis of a Boranophoshate Dimer,(R_(P))-Ammonium thymidin-3′-yl thymidin-5′-yl boranophosphate[(R_(P))-7tt] via Route B

This compound was obtained by using “L-2 (52 μmol)” instead of “D-2 (52μmol)” in a similar manner to Example 98. The product was identical tothat of a control sample synthesized by the conventional H-phosphonatemethod. The yield of (R_(P))-7tt was determined by a UV absorbancemeasurement at 260 nm with the molar extinction coefficient of anapproximate value for natural T1′ dimer (16800). The yield of(R_(P))-7tt was 90% yield, R_(P):S_(P)=95:5. Retention time: 9.8 min((S_(P))-7tt: 9.7 min). The UPLC profile is shown in FIG. 26.

Example 100 Solid-Phase Synthesis of aN-[(2-dimethylamino)ethyl]phosphoramidate Dimer, (S_(P))-Thymidin-3′-ylthymidin-5′-yl N-[(2-dimethylamino)ethyl]phosphoramidate [(S_(P))-8tt]via Route B

N³-Benzoyl-5′-O-(4,4′-dimethoxytrityl)thymidine-loaded HCP resin (16.4mg; 30.5 μmol/g, 0.5 μmol) via a succinyl linker was treated with 1%TFA/DCM (3×1 mL) for the removal of the 5′-O-DMTr group, washed with DCM(3×1 mL) and dry MeCN (3×1 mL), and dried in vacuo. Pre-activatedmonomer solution (200 μL, 50 μmol; which consists of8-diazabicyclo[5.4.0]undec-7-eniumN³-benzoyl-5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphonate (50μmol, for H-phosphonate monoester), MeCN-CMP (9:1, v/v, for solvent),BTC (32 μmol, for condensing reagent), and L-6 (52 μmol, foraminoalcohol)) was added followed by the addition of 5 M CMPT/MeCN (50μL, 250 μmol). Being stirred for 2 min, the reaction solution wasremoved, and the resin was washed with MeCN (3×1 mL), DCM (3×1 mL), anddried under the reduced pressure (>5 min). The 5′-O-DMTr group and thechiral auxiliary was simultaneously removed by treatment with 1% TFA inDCM (3×1 mL), and the resin was washed with DCM (3×1 mL) and dry MeCN(3×1 mL), and dried in vacuo. The resulting intermediate on the resinwas amidated by treatment with a mixture of CCl₄-Me₂N(CH₂)₂NH₂ (1 mL,1:9, v/v) for 30 min, the resin was then washed with DCM (3×1 mL). Thephosphoramidate dimer on the resin was then treated with 2 M NH₃/EtOH (2mL) for 12 h at room temperature to remove the protecting groups of thenucleobases and also to release the dimer from the resin. The resin wasremoved by filtration and washed with MeOH. The filtrate wasconcentrated to dryness. The residue was dissolved in H₂O (2 mL), washedwith Et₂O (3×2 mL), and the combined washings were back-extracted withH₂O (2 mL). The combined aqueous layers were concentrated to dryness.The resulting crude product was analyzed by reversed-phase UPLC® with alinear gradient of 0-20% MeOH in 0.1 M ammonium acetate buffer (pH 7.0)for 15 min at 55° C. at a rate of 0.4 ml/min. The product was identicalto that of a control sample synthesized by the conventionalH-phosphonate method. The yield of (S_(P))-8tt was determined by a UVabsorbance measurement at 260 nm with the molar extinction coefficientof an approximate value for natural TT dimer (16800). The yield of(S_(P))-8tt was 90% (R_(P):S_(P)=6:94). Retention time: 10.3 min((R_(P))-8tt: 9.6 min). The UPLC profile is shown in FIG. 27.

Example 101 Solid-Phase Synthesis of aN-[(2-dimethylamino)ethyl]phosphoramidate Dimer, (R_(P))-Thymidin-3′-ylthymidin-5′-yl N-[(2-dimethylamino)ethyl]phosphoramidate [(R_(P))-8tt]via Route B

This compound was obtained by using “D-2 (52 μmol)” instead of “L-2 (52μmol)” in a similar manner to Example 100. The product was identical tothat of a control sample synthesized by the conventional H-phosphonatemethod. The yield of (R_(P))-8tt was determined by a UV absorbancemeasurement at 260 nm with the molar extinction coefficient of anapproximate value for natural TT dimer (16800). The yield of (R_(P))-8ttwas 86% yield, R_(P):S_(P)=96:4. Retention time: 9.6 min ((S_(P))-8tt:10.3 min). The UPLC profile is shown in FIG. 28.

Example 102 Solid-Phase Synthesis of a Phosphorothioate Tetramer,All-(S_(P))-[T_(PS)]₃T (Phosphorothioate) via Route A

5′-O-(4,4′-dimethoxytrityl)thymidine-loaded HCP resin (0.5 μmol) via asuccinyl linker was used for the synthesis. Repeating the steps in Table3 performs chain elongation. After the chain elongation, the 5′-O-DMTrgroup was removed by treatment with 3% DCA/DCM (3×1 mL), and washed withDCM (3×1 mL). The phosphorothioate tetramer on the resin was thentreated with 25% NH₃ for 12 h at 55° C. to remove the chiral auxiliariesand the protecting groups of the nucleobases and also to release thetetramer from the resin. The resin was removed by filtration and washedwith H₂O. The filtrate was concentrated to dryness. The residue wasdissolved in H₂O (2 mL), washed with Et₂O (3×2 mL), and the combinedwashings were back-extracted with H₂O (2 mL). The combined aqueouslayers were concentrated to dryness. The resulting crude product wasanalyzed by reversed-phase UPLC® with a linear gradient of 0-30% MeOH in0.1 M ammonium acetate buffer (pH 7.0) for 30 min at 55° C. at a rate of0.4 ml/min. The product was identical to that of a control samplesynthesized by the conventional phosphoramidite method. The yield of theproduct was determined by a UV absorbance measurement at 260 nm with themolar extinction coefficient of an approximate value for natural T₄tetramer (33000). Average coupling yield was 96%, optical purity was 96%(average: 99%). Retention time: 23.0 min; MS (MALDI TOF-MS) m/z Calcdfor C₄₀H₅₂N₈O₂₃P₃S₃ [M−H]⁻ 1201.15, found 1200.97. The UPLC profile isshown in FIG. 29.

TABLE 3 step operation reagents and solvents time 1 detritylation 3%DCA/DCM 3 × 30 s 2 washing (i) DCM (ii) dry MeCN (iii) — drying invacuo. 3 coupling 5M CMPT/MeCN (50 μL, 5 min 250 μmol) pre-activated(R_(P))- or (S_(P))-monomer solution (200 μL, 25 μmol)* 4 washing (i)MeCN (ii) drying in vacuo. — 5 capping (i) 0.5M CF₃COIm/dry THF 30 s(ii) 1M DMAN/dry THF 6 washing (i) dry MeCN (ii) drying in vacuo. — 7sulfuryzation 0.3M DTD/MeCN 5 min 8 washing (i) MeCN (ii) DCM —

* Preparation of “pre-activated (R_(P))- or (S_(P))-monomer solution”

8-Diazabicyclo[5.4.0]undec-7-eniumN³-benzoyl-5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphonate (25μmol) was dried by repeated coevaporations with dry pyridine and drytoluene, then dissolved in dry MeCN-CMP (9:1, v/v). To the solution,Ph₃PCl₂ (62.5 μmol) was added, and stirred for 10 min. L-2 (30 μmol; D-2for “S_(P)” solution) was then added and stirred for additional 10 minto give pre-activated monomer solution.

Example 103 Solid-Phase Synthesis of a Phosphorothioate Tetramer,(S_(P), R_(P), S_(P))-[T_(PS)]₃T (Phosphorothioate) via Route A

This compound was obtained in a similar manner to All-(S_(P))-[T_(PS)]₃Tin Example 102. The yield of the product was determined by a UVabsorbance measurement at 260 nm with the molar extinction coefficientof an approximate value for natural T₄ tetramer (33000). The product wasidentical to that of a control sample synthesized by the conventionalphosphoramidite method. Average coupling yield is 96%, optical purity is94% (average: 98%). Retention time: 22.3 min; MS (MALDI TOF-MS) m/zCalcd for C₄₀H₅₂N₈O₂₃P₃S₃ [M−H]⁻ 1201.15, found 1200.97. The UPLCprofile is shown in FIG. 30.

Example 104 Solid-Phase Synthesis of a Phosphorothioate Tetramer,(R_(P), S_(P), R_(P))-[T_(PS)]₃T (Phosphorothioate) via Route A

This compound was obtained in a similar manner to All-(S_(P))-[T_(PS)]₃Tin Example 102. The yield of the product was determined by a UVabsorbance measurement at 260 nm with the molar extinction coefficientof an approximate value for natural T₄ tetramer (33000). The product wasidentical to that of a control sample synthesized by the conventionalphosphoramidite method. Average coupling yield is 97%, optical purity is96% (average: 99%). Retention time: 21.7 min; MS (MALDI TOF-MS) m/zCalcd for C₄₀H₅₂N₈O₂₃P₃S₃ [M−H]⁻ 1201.15, found 1200.96. The UPLCprofile is shown in FIG. 31.

Example 105 Solid-Phase Synthesis of a Phosphorothioate Tetramer,All-(R_(P))-[T_(PS)]₃T (Phosphorothioate) via Route A

This compound was obtained in a similar manner toAll-(S_(P))-[T_(PS)]₃T. The yield of the product was determined by a UVabsorbance measurement at 260 nm with the molar extinction coefficientof an approximate value for natural T₄ tetramer (33000). The product wasidentical to that of a control sample synthesized by the conventionalphosphoramidite method. Average coupling yield is 95%, optical purity is92% (average: 97%). Retention time: 19.1 min; MS (MALDI TOF-MS) m/zCalcd for C₄₀H₅₂N₈O₂₃P₃S₃ [M−H]⁻ 1201.15, found 1200.92. The UPLCprofile is shown in FIG. 32.

Example 106 Solid-Phase Synthesis of a RNA Phosphorothioate Tetramer,(S_(P))-Ammonium 2′-O-methyluridin-3′-yl uridin-5′-yl phosphorothioate[(S_(P))-9u_(M)u] via Route A

2′-O-Acetyl-N³-benzoyl-5′-O-(4,4′-dimethoxytrityl)uridine-loaded CPGresin (21.4 mg; 23.4 μmol/g, 0.5 μmol) via a succinyl linker was treatedwith 3% DCA/DCM (3×1 mL), then washed with DCM (3×1 mL) and dry MeCN(3×1 mL). After the resin was dried under the reduced pressure (>5 min),pre-activated monomer solution (250 μL, 25 μmol; which consists of8-diazabicyclo[5.4.0]undec-7-eniumN³-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-methyluridin-3′-ylphosphonate (25 μmol, for H-phosphonate monoester), MeCN-pyridine (9:1,v/v, for solvent), Ph₃PCl₂ (62.5 μmol, for condensing reagent), and L-2(30 μmol, for aminoalcohol)) was added. Being stirred for 5 min, thereaction solution was removed, and the resin was washed with MeCN (3×1mL), dried under the reduced pressure (>5 min). The resultingintermediate was sulfurized by treatment with 0.3 M DTD/MeCN (500 μL,150 μmol) for 5 min, then washed with MeCN (3×1 mL) and DCM (3×1 mL).The 5′-O-DMTr group was removed by treatment with 3% DCA/DCM (3×1 mL),and washed with DCM (3×1 mL). The phosphorothioate dimer on the resinwas then treated with 25% NH₃ (1 mL) for 12 h at 55° C. to remove thechiral auxiliary and the protecting groups of the nucleobases and alsoto release the dimer from the resin. The resin was removed by filtrationand washed with H₂O. The filtrate was concentrated to dryness. Theresidue was dissolved in H₂O (2 mL), washed with Et₂O (3×2 mL), and thecombined washings were back-extracted with H₂O (2 mL). The combinedaqueous layers were concentrated to dryness. The resulting crude productwas analyzed by reversed-phase UPLC® with a linear gradient of 0-20%MeOH in 0.1 M ammonium acetate buffer (pH 7.0) for 15 min at 55° C. at arate of 0.4 ml/min. The yield of (S_(P))-9u_(M)u was 95%(R_(P):S_(P)=2:98). Retention time: 10.2 min ((R_(P))-9u_(M)u: 9.3 min);MS (MALDI TOF-MS) m/z Calcd for C₁₉H₂₄N₄O₁₃PS [M−H]⁻ 579.08, found578.92. The UPLC profile is shown in FIG. 33.

Example 107 Solid-Phase Synthesis of a RNA Phosphorothioate Tetramer,(R_(P))-Ammonium 2′-O-methyluridin-3′-yl uridin-5′-yl phosphorothioate[(R_(P))-9u_(M)u] via Route A

This compound was obtained by using “D-2 (30 μmol)” instead of “L-2 (30μmol)” in a similar manner to Example 106. The yield of (R_(P))-9u_(M)uwas 94% (R_(P):S_(P)=95:5). Retention time: 9.3 min ((S_(P))-9u_(M)u:10.3 min); MS (MALDI TOF-MS) m/z Calcd for C₁₉H₂₄N₄O₁₃PS [M−H]⁻ 579.08,found 578.97. The UPLC profile is shown in FIG. 34.

Example 108 Solid-Phase Synthesis of a RNA Phosphorothioate Tetramer,(S_(P))-Ammonium 2′-deoxy-2′-fluorouridin-3′-yl uridin-5′-ylphosphorothioate [(S_(P))-10u_(F)u] via Route A

This compound was obtained by using “8-diazabicyclo[5.4.0]undec-7-eniumN³-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxy-2′-fluorouridin-3′-ylphosphonate (25 μmol)” instead of “8-diazabicyclo[5.4.0]undec-7-eniumN³-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-methyluridin-3′-ylphosphonate (25 μmol)” in a similar manner to Example 106. The yield of(S_(P))-10u_(F)u was 93% (R_(P):S_(P)=1:99). Retention time: 10.6 min((R_(P))-10u_(F)u: 8.5 min); MS (MALDI TOF-MS) m/z Calcd forC₁₈H₂₁FN₄O₁₂PS [M−H]⁻ 567.06, found 566.96. The UPLC profile is shown inFIG. 35.

Example 109 Solid-Phase Synthesis of a RNA Phosphorothioate Tetramer,(R_(P))-Ammonium 2′-deoxy-2′-fluorouridin-3′-yl uridin-5′-ylphosphorothioate [(R_(P))-10u_(F)u] via Route A

This compound was obtained by using “D-2 (30 μmol)” instead of “L-2 (30μmol)” in a similar manner to Example 108. The yield of (R_(P))-10u_(F)uwas 92% yield, R_(P):S_(P)=96:4. Retention time: 8.4 min((S_(P))-10u_(F)u: 10.7 min); MS (MALDI TOF-MS) m/z Calcd forC₁₈H₂₁FN₄O₁₂PS [M−H]⁻ 567.06, found 566.97. The UPLC profile is shown inFIG. 36.

Example 110 Solution-Phase Synthesis of Unnatural Nucleobase(S_(P))-Ammonium 1-(3-nitropyrrol-1-yl)-2-deoxyribofuranos-3-ylthymidin-5′-yl phosphorothioate [(S_(P))-11nt] via Route A

This compound was obtained by using “8-diazabicyclo[5.4.0]undec-7-enium5-O-(4,4′-dimethoxytrityl)-1-(3-nitropyrrol-1-yl)-2-deoxyribofuranos-3-ylphosphonate (25 μmol)” instead of “8-diazabicyclo[5.4.0]undec-7-eniumN³-benzoyl-5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphonate (25μmol)” in a similar manner to Example 78. The yield of the product wasdetermined by a UV absorbance measurement at 260 nm with the molarextinction coefficient of an approximate value for natural CT dimer(15200). The yield of (S_(P))-11nt was 98% yield. Retention time: 16.3min. (R_(P))-11nt could not be resolved; MS (MALDI TOF-MS) m/z Calcd forC₁₉H₂₄N₄O₁₁PS [M−H]⁻ 547.09, found 547.02. The UPLC profile is shown inFIG. 37.

Example 111 Solution-Phase Synthesis of Unnatural Nucleobase(R_(P))-Ammonium 1-(3-nitropyrrol-1-yl)-2-deoxyribofuranos-3-ylthymidin-5′-yl phosphorothioate [(R_(P))-11 nt] via Route A

This compound was obtained by using “D-2 (30 μmol)” instead of “L-2 (30μmol)” in a similar manner to Example 110. The yield of the product wasdetermined by a UV absorbance measurement at 260 nm with the molarextinction coefficient of an approximate value for natural CT dimer(15200). The yield of (R_(P))-11nt was 97% yield. Retention time: 16.1min. (S_(P))-11nt could not be resolved; MS (MALDI TOF-MS) m/z Calcd forC₁₉H₂₄N₄O₁₁PS [M−H]⁻ 547.09, found 547.01. The UPLC profile is shown inFIG. 38.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A method for a synthesis of a nucleic acid comprising a chiralX-phosphonate moiety comprising: reacting a molecule comprising anachiral H-phosphonate moiety, a chiral reagent, and a nucleosidecomprising a 5 ′-OH moiety to form a condensed intermediate; andconverting the condensed intermediate to the nucleic acid comprising achiral X-phosphonate moiety; wherein: the nucleic acid comprising achiral X-phosphonate moiety is a compound of Formula 1:

wherein: R¹ is —OH or —OR^(a); R^(a) is a blocking moiety; each instanceof R² is independently hydrogen, —OH, halogen, or —OR^(b); R^(b) is ablocking moiety; each instance of Ba is independently a blocked orunblocked adenine, cytosine, guanine, thymine, uracil or modifiednucleobase; each instance of X is independently —S⁻Z⁺; Z⁺ is an ammoniumion, alkylammonium ion, heteroaromatic iminium ion, or heterocycliciminium ion, any of which is primary, secondary, tertiary or quaternary,or Z⁺ is a monovalent metal ion; R³ is hydrogen, a blocking group, alinking moiety connected to a solid support or a linking moietyconnected to a nucleic acid; and n is an integer of 1 to about
 200. 2.The method of claim 1, wherein the molecule comprising an achiralH-phosphonate moiety is a compound of Formula 2:

wherein: R¹ is —OH or —OR^(a); R^(a) is a blocking moiety; R² ishydrogen, —OH, halogen, or —OR^(b); R^(b) is a blocking moiety; Baindependently a blocked or unblocked adenine, cytosine, guanine,thymine, uracil or modified nucleobase; and Z⁺ is an ammonium ion,alkylammonium ion, heteroaromatic iminium ion, or heterocyclic iminiumion, any of which is primary, secondary, tertiary or quaternary, or Z⁺is a monovalent metal ion.
 3. The method of claim 2, wherein the chiralreagent is a compound of Formula 3:

wherein: W₁ is —NG⁵-; W₂ is —O—; G¹, G², G³, G⁴, and G⁵ areindependently hydrogen, alkyl, aralkyl, cycloalkyl, cycloalkylalkyl,heterocyclyl, hetaryl, or aryl, or two of G¹, G², G³, G⁴, and G⁵ are G⁶which are taken together to form a saturated, partially unsaturated orunsaturated carbocyclic or heteroatom-containing ring of up to about 20ring atoms which is monocyclic or polycyclic, fused or unfused, andwherein no more than four of G¹, G², G³, G⁴, and G⁵ are G⁶.
 4. Themethod of claim 3, wherein the nucleoside comprising a 5′-OH moiety is acompound of Formula 4:

wherein: each instance of R² is independently hydrogen, halogen, or—OR^(b); R^(b) is a blocking moiety; each instance of Ba isindependently a blocked or unblocked adenine, cytosine, guanine,thymine, uracil or modified nucleobase; m is an integer of 0 to n−1;O_(A) is connected to a linking moiety connected to a solid support or alinking moiety connected to a nucleic acid; J is O and D is H; or J isS, and D is a moiety of Formula A:

wherein: W₁ and W₂ are independently NG⁵, O, or S; A is hydrogen, acyl,aryl, alkyl, aralkyl, or silyl moiety; and wherein G¹, G², G³, G⁴, andG⁵ are independently hydrogen, alkyl, aralkyl, cycloalkyl,cycloalkylalkyl, heterocyclyl, heteroaryl, or aryl, or two of G¹, G²,G³, G⁴, and G⁵ are G⁶ which are taken together to form a saturated,partially unsaturated or unsaturated carbocyclic orheteroatom-containing ring of up to about 20 ring atoms which ismonocyclic or polycyclic, fused or unfused and wherein no more than fourof G¹, G², G³, G⁴, and G⁵ are G⁶.
 5. The method of claim 4, wherein thestep of converting the condensed intermediate to a compound of Formula 1comprises: capping the condensed intermediate and modifying the cappedcondensed intermediate to produce a compound of Formula 5:

wherein: R¹ is —OH or —OR^(a); R^(a) is a blocking moiety; each instanceof R² is independently hydrogen, —OH, halogen, or —OR^(b); R^(b) is ablocking moiety; each instance of Ba is independently a blocked orunblocked adenine, cytosine, guanine, thymine, uracil or modifiednucleobase; each instance of J is S; v is an integer of 2 to n−1; O_(A)is connected to a linking moiety connected to a solid support or alinking moiety connected to a nucleic acid; A is an acyl, aryl, alkyl,aralkyl, or silyl moiety; and W₁ and W₂ are independently NG⁵, O, or S;and G¹, G², G³, G⁴, and G⁵ are independently hydrogen, alkyl, aralkyl,cycloalkyl, cycloalkylalkyl, heterocyclyl, heteroaryl, or aryl, or twoof G¹, G², G³, G⁴, and G⁵ are G⁶ which are taken together to form asaturated, partially unsaturated or unsaturated carbocyclic orheteroatom-containing ring of up to about 20 ring atoms which ismonocyclic or polycyclic, fused or unfused and wherein no more than fourof G¹, G², G³, G⁴, and G⁵ are G⁶.
 6. The method of claim 5, wherein ineach of formulae 3-5, G¹, G², and G³ are independently hydrogen, alkyl,aralkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, hetaryl, or aryl,and G⁴ and G⁵ are taken together to form a saturated or partiallyunsaturated heteroatom-containing ring of up to about 20 ring atomswhich is monocyclic or polycyclic, fused or unfused.
 7. The method ofclaim 6, wherein G⁴ and G⁵ are taken together to form a saturated5-membered saturated ring containing one nitrogen atom.
 8. The method ofclaim 7, wherein one of G¹ and G² is alkyl, aralkyl, cycloalkyl,cycloalkylalkyl, heterocyclyl, hetaryl, or aryl.
 9. The method of claim8, wherein the other of G¹ and G² is alkyl, aralkyl, cycloalkyl,cycloalkylalkyl, heterocyclyl, hetaryl, or aryl.
 10. The method of claim8, wherein the other of G¹ and G² is hydrogen.
 11. The method of claim9, wherein G³ is hydrogen.
 12. The method of claim 11, wherein thechiral reagent of formula 3 is


13. The method of claim 10, wherein G³ is hydrogen.
 14. The method ofclaim 13, wherein the chiral reagent of formula 3 is


15. The method of claim 6, further comprising providing an activatingreagent.
 16. The method of claim 15, wherein the activating reagent isimidazole, 4,5-dicyanoimidazole (DCI), 4,5-dichloroimidazole,1-phenylimidazolium triflate (PhIMT), benzimidazolium triflate (BIT),benztriazole, 3-nitro-1,2,4-triazole (NT), tetrazole,5-ethylthiotetrazole, 5-(4-nitrophenyl)tetrazole,N-cyanomethylpyrrolidinium triflate (CMPT), N-cyanomethylpiperidiniumtriflate, or N-cyanomethyldimethylammonium triflate.
 17. The method ofclaim 5, wherein R^(a) is substituted or unsubstituted trityl orsubstituted silyl.
 18. The method of claim 17, wherein R^(b) issubstituted or unsubstituted methyl ether.
 19. The method of claim 18,wherein R³ is a linking moiety connected to a solid support or a linkingmoiety connected to a nucleic acid.
 20. The method of claim 5, wherein nis about 10 to about 200.